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Partial purification of serum factor that causes necrosis of tumors
Abstract
Tumor necrosis can be induced in transplanted mouse methylcholanthrene-induced sarcoma by a tumor necrosis factor in the serum of mice infected with bacillus Calmette-Guérin and given bacterial endotoxin. Sera from normal mice, endotoxin-treated mice, and mice infected with bacillus Calmette-Guérin do not contain this factor. A 20- to 30-fold purification of the serum factor has been achieved by (NH4)2SO4 fractionation, Sephadex G-100 and G-200 gel filtration, and preparative polyacrylamide electrophoresis. Tumor necrosis factor is not bacterial endotoxin. It migrates with alpha-globulins, is made up of at least four subunits, and has a molecular weight of about 150,000. The active factor is a glycoprotein that contains sialic acid and galactosamine.
... Tumour necrosis factor (TNF)-a is an important cytokine implicated in the pathogenesis of juvenile idiopathic arthritis (JIA), and can induce expression of other proinflammatory cytokines such as interleukin (IL)-1b, IL-6 and IL-8, leading to an overall protracted inflammatory response [1][2][3][4]. In the normal physiological state, proinflammatory cytokines, including TNF-a, are maintained in equilibrium with anti-inflammatory cytokines, such as IL-10. ...
... HC subjects were recruited from orthopaedic clinics if preoperative blood work was to be drawn, and were excluded if they had any medical condition suspected to alter cytokine levels. HCs were also excluded if they [1] had a physician-diagnosed infection (with or without antibiotic treatment), [2] received immunizations within 1 month prior to enrolment or [3] had a fever within 2 weeks prior to enrolment. Similarly, cytokine levels were not drawn for JIA subjects if they [1] had a physiciandiagnosed infection (with or without antibiotic treatment), [2] received immunizations within 1 month prior to a visit or [3] had a fever within 2 weeks prior to a visit. ...
... HCs were also excluded if they [1] had a physician-diagnosed infection (with or without antibiotic treatment), [2] received immunizations within 1 month prior to enrolment or [3] had a fever within 2 weeks prior to enrolment. Similarly, cytokine levels were not drawn for JIA subjects if they [1] had a physiciandiagnosed infection (with or without antibiotic treatment), [2] received immunizations within 1 month prior to a visit or [3] had a fever within 2 weeks prior to a visit. Subjects with primary or acquired immunodeficiencies were excluded from both groups. ...
OBJECTIVE:
To prospectively evaluate cytokine levels and disease activity in juvenile idiopathic arthritis (JIA) patients treated with and without tumor necrosis factor (TNF)-α inhibitors.
METHODS:
TNF-α inhibitor-naïve JIA subjects were followed prospectively over 6 months. Cytokine levels of TNF-α, interleukin (IL)-1β, IL-6, IL-8, IL-10 and IL-17 were measured at baseline for JIA subjects and healthy controls (HCs). Cytokine levels were then measured at 4 time points after initiation of TNF-α inhibition for anti-TNF-α treated (anti-TNF) JIA subjects, and at 2 subsequent time points for other JIA (non-TNF) subjects. JIA disease activity by Childhood Health Assessment Questionnaire (CHAQ) disability index/pain score and physician joint count/global assessment was recorded.
RESULTS:
Sixteen anti-TNF, 31 non-TNF, and 16 HCs were analyzed. Among JIA subjects, those with higher baseline disease activity (subsequent anti-TNFs) had higher baseline TNF-α, IL-6 and IL-8 than those with lower disease activity (non-TNFs) (p <0.05). TNF-α and IL-10 increased, and IL-6 and IL-8 no longer remained significantly higher after TNF-α inhibitor initiation in anti-TNF subjects. Subgroup analysis of etanercept versus adalimumab treated subjects showed that TNF-α and IL-17 increased significantly in etanercept but not adalimumab treated subjects, despite clinical improvement in both groups of subjects.
CONCLUSION:
JIA subjects with increased disease activity at baseline had higher serum pro-inflammatory cytokines. TNF-α inhibition resulted in suppression of IL-6 and IL-8 in parallel with clinical improvement in all anti-TNF treated subjects, but also was associated with elevated TNF-α and IL-17 in etanercept treated subjects.
... TNF was originally identified as a glycoprotein found in the serum of Bacillus Calmette-Guérin-infected infected mice given endotoxin (also known as lipopolysaccharide). When this serum was administered intravenously, necrosis of sarcomas that had been transplanted subcutaneously into mice was observed 29,30 . Cloning of human TNF was reported 10 years later by several groups 31,32 . ...
... Antitumor activities of tumor necrosis factor As mentioned above, TNF was originally identified as a glycoprotein found in the serum of Bacillus Calmette-Guérin-infected mice given endotoxin (also known as lipopolysaccharide). This serum could induce necrosis of sarcomas that had been transplanted subcutaneously into mice when administered intravenously 29,30 . The structurally related protein LT has also demonstrated tumor necrosis activity 39 . ...
Objective:
Tumor necrosis factor (TNF) is a highly pleiotropic cytokine with multiple activities other than its originally discovered role of tumor necrosis in rodents. TNF is now understood to play a contextual role in driving either tumor elimination or promotion. Using both animal and human data, this review examines the role of TNF in cancer development and the effect of TNF and TNF inhibitors (TNFis) on malignancy risk.
Research design:
A literature review was performed using relevant search terms for TNF and malignancy.
Results:
Although administration of TNF can cause tumor regression in specific rodent tumor models, human expression polymorphisms suggest that TNF can be a tumor-promoting cytokine, whereas blocking the TNF pathway in a variety of tumor models inhibits tumor growth. In addition to direct effects of TNF on tumors, TNF can variously affect immunity and the tumor microenvironment. Whereas TNF can promote immune surveillance designed to eliminate tumors, it can also drive chronic inflammation, autoimmunity, angiogenesis, and other processes that promote tumor initiation, growth, and spread. Key players in TNF signaling that shape this response include NF-κB and JNK, and malignant-inflammatory cell interactions, each of which may have different responses to TNF signaling. Focusing on rheumatoid arthritis (RA) patients, where clinical experience is most extensive, a review of the clinical literature shows no increased risk of overall malignancy or solid tumors such as breast and lung cancers with exposure to TNFis. Lymphoma rates are not increased with use of TNFis. Conflicting data exist regarding the risks of melanoma and nonmelanoma skin cancer. Data regarding the risk of recurrent malignancy are limited.
Conclusions:
Overall, the available data indicate that elevated TNF is a risk factor for cancer, whereas its inhibition in RA patients is not generally associated with an increased cancer risk. In particular, TNF inhibition is not associated with cancers linked to immune suppression. A better understanding of the tumor microenvironment, molecular events underlying specific tumors, and epidemiologic studies of malignancies within specific disease indications should enable more focused pharmacovigilance studies and a better understanding of the potential risks of TNFis.
... The background activity of NMS can partly be ascribed to the presence of minor amounts of TNF (Shah, Green & Moore, 1978; Green, Chiasson & Shah, 1979) and partly to another factor shown to inhibit DNA synthesis by mouse lymphocytes (Nelson & Shneider, 1974; Smith & HammarstrOm, 1979 ). This factor shares some physicochemical characteristics with TNF such as its nondialysable glycoprotein nature, its heat-stability (30 min, 56°C) and its electrophoretic mobility as an a-globulin (Nelson & Shneider, 1974; Green, Dobrjansky, Carswell, Kassel, Old, Fiore & Schwartz, 1976). TNF, however, has another elution pattern on Sephadex G20 o (Green et al., 1976) and unlike the other inhibitory factor (Nelson, 1972), cannot be In vitro Anti-tumour Activity demonstrated nor induced in serum of nude mice (Hoffmann, Oettgen, Old, Mittler & Hammerling, 1978). ...
... This factor shares some physicochemical characteristics with TNF such as its nondialysable glycoprotein nature, its heat-stability (30 min, 56°C) and its electrophoretic mobility as an a-globulin (Nelson & Shneider, 1974; Green, Dobrjansky, Carswell, Kassel, Old, Fiore & Schwartz, 1976). TNF, however, has another elution pattern on Sephadex G20 o (Green et al., 1976) and unlike the other inhibitory factor (Nelson, 1972), cannot be In vitro Anti-tumour Activity demonstrated nor induced in serum of nude mice (Hoffmann, Oettgen, Old, Mittler & Hammerling, 1978). Cell density also influenced the results of the test (Fig. 2). ...
A method measuring 3H-thymidine incorporation in Meth A sarcoma cells was used to quantify in vitro anti-tumour activity of tumour necrosis serum and compared with a method using cell viability as a parameter. Tumour necrosis serum obtained from mice pretreated with Corynebacterium parvum and elicited with endotoxin two weeks later greatly inhibited 3H-thymidine incorporation, whereas sera of normal mice and of mice treated with C. parvum or endotoxin alone were much less inhibitory. All sera reduced viable cell numbers, tumour necrosis serum being most active.The 3H-thymidine incorporation assay is suited for screening mouse sera on anti-tumour activity. It was shown that the anti-tumour activity of normal mouse serum can be potentiated by in vivo pretreatment of mice with bacterial agents. The mechanism of the anti-tumour action(s) and the factor(s) involved remain to be elucidated.
... Other cytokines synthesized by keratinocytes or stromal cells are likely to control the growth and fibroplasia of epithelial-stromal (vascular) in psoriatic lesions. TGF-β, IL-1, IL-6, and IL-20 may act as autocrine and/or paracrine keratinocyte growth factors [32]. ...
Psoriasis is a common, chronic systemic inflammatory disease affecting 125 million people worldwide. It is associated with several important conditions, including psoriatic arthritis, cardiometabolic syndrome, and depression, leading to a significant reduction in patients’ quality of life. Current treatments only reduce symptoms, not cure. This review discusses the mechanisms involved in the initiation and development of the disease, the role of oxidative stress in this autoimmune disease, as well as potential therapeutic options with substances of natural origin. The main aim of the study is intended to offer a review of the literature to present plants and phytochemicals that can represent potential remedies in the fight against psoriasis. We identified many in vitro, in vivo, and clinical trials studies that evaluated the relationship between chosen natural substances and immune system response in the course of psoriasis. We sought to find articles about the efficacy of potential natural-derived drugs in controlling symptoms and their ability to maintain long-term disease inactivity without side effects, and the result of our work is a review, which highlights the effectiveness of plant-derived drugs in controlling the inflammatory burden on psoriatic patients by decreasing the oxidative stress conditions.
... U takto rozdílných mikroorganizmů by bylo namístě Rozuzlení přišlo v polovině 70. let minulého století, kdy byl při zkoumání protinádorových imunitních mechanizmů popsán kachektin, nyní známý spíše pod akro nymem TNFα (tumor necrosis factor α). V prvních publikacích byl představen jako glykoproteinový mediátor, který produkují makrofágy po kontaktu s patogenními bakteriemi [4,5]. Poté vyšlo postupně najevo, že tento mediátor je hlavním aktivátorem pyogenní zánětlivé reakce, podporuje vznik horečky, je odpovědný za zapojení endoteliálních buněk do zánětlivé odpovědi a rovněž indukuje proteokatabolizmus a další metabolické změny, které sepsi provázejí. ...
... TNF-α is a major proinflammatory cytokine produced primarily during early inflammatory events. Mainly known to be secreted by activated macrophages and monocytes, TNF-α can also be produced by other immune cell types including activated T cells, NK cells, mast cells, as well as by non-immune cells such as endothelial cells and fibroblasts in varying proportions [332][333][334][335]. TNF-α can bind to two membrane bound receptors, TNFR1 and TNFR2. ...
Tumors represent a hostile environment for the effector cells of cancer immunosurveillance. Immunosuppressive receptors and soluble or membrane-bound ligands are abundantly exposed and released by malignant entities and their stromal accomplices. As a consequence, executioners of antitumor immunity inefficiently navigate across cancer tissues and fail to eliminate malignant targets. By inducing immunogenic cancer cell death, oncolytic viruses profoundly reshape the tumor microenvironment. They trigger the local spread of danger signals and tumor-associated (as well as viral) antigens, thus attracting antigen-presenting cells, promoting the activation and expansion of lymphocytic populations, facilitating their infiltration in the tumor bed, and reinvigorating cytotoxic immune activity. The present review recapitulates key chemokines, growth factors and other cytokines that orchestrate this ballet of antitumoral leukocytes upon oncolytic virotherapy.
... TNF was originally described as "a soluble factor found in sera from mice that were sequentially treated with a reticulo-endothelial stimulator {Mycobacterium bovis, strain BCG) and bacterial endotoxin or LPS" (Gifford and Flick, 1987). TNF was isolated from rabbit and human sera (Matthews, 1978;Green et al. 1976) and in 1984 the TNF gene was cloned (Pennica et al. 1984). Soon after, the proteins amino acid sequence was identified and recombinant TNF produced Pennica et al. 1985;Shirai et al. 1985). ...
The influence of tumour relevant oxygen tensions on the cytotoxic action of TNFα on tumour cells, both in vitro and in vivo, was investigated. Tumour oxygen tensions were assessed in two murine tumours (SaF and CaNT), directly, using the Eppendorf pO2 histograph, and indirectly, by determining the radiobiological hypoxic fraction. These tumours are largely hypoxic with median pO2 values less than 2% oxygen (15 mmHg). Such oxygen tensions re-established in vitro induce resistance to TNFα cytotoxicity in murine and human tumour cell lines. The mechanism(s) of hypoxia-induced resistance to TNFα cytotoxicity was studied using the SaF cell line. The role of cell cycle was assessed but was found to be negligible. Western blotting and ELISA techniques were used to assess putative protective proteins such as manganese superoxide dismutase (MnSOD) and TNFα itself. MnSOD is not implicated in hypoxia-induced TNF-resistance. Endogenous TNFα levels were assessed and found to be significantly induced during hypoxic preincubation. Indeed, both pre-treatment of oxic cells with exogenous TNFα and overexpression of endogenous TNFα, by gene transfection, induced resistance to TNFα challenge. A growth delay assay and an in vivo/in vitro clonogenic assay determined that the direct cytotoxic action of TNFα in vivo is not the only mechanism implicated in its antitumour activity. Indeed, TNFα significantly reduces tumour blood flow, as demonstrated by laser Doppler flowmetry, and this may be largely responsible for the antitumour action of TNFα in vivo. The cytotoxic action of TNFα is dramatically modulated by oxygen tensions known to exist in tumours. Thus, overcoming tumour hypoxia could potentially increase TNFα cytotoxicity and together with the effective anti-vascular effects could perhaps improve the antitumour efficacy of TNFα.
... TNFα was named due to original research that in 1975 determined that TNFα causes hemorrhagic necrosis of tumors when found in high concentration (2)(3)(4). Later, it was discovered that TNFα was involved in a plethora of cellular processes and, more importantly, that it had paradoxical effects. It was initially described that TNFα was mainly produced by activated macrophages, monocytes, NK cells, T lymphocytes, neutrophils and mast cells, but afterwards it was discovered that it was also expressed by non-immune cells like fibroblasts, endothelial cells, cardiac myocytes and neurons, among others (5,6). ...
Breast cancer is the most frequently diagnosed cancer and the principal cause of mortality by malignancy in women and represents a main problem for public health worldwide. Tumor necrosis factor α (TNFα) is a pro-inflammatory cytokine whose expression is increased in a variety of cancers. In particular, in breast cancer it correlates with augmented tumor cell proliferation, higher malignancy grade, increased occurrence of metastasis and general poor prognosis for the patient. These characteristics highlight TNFα as an attractive therapeutic target, and consequently, the study of soluble and transmembrane TNFα effects and its receptors in breast cancer is an area of active research. In this review we summarize the recent findings on TNFα participation in luminal, HER2-positive and triple negative breast cancer progression and metastasis. Also, we describe TNFα role in immune response against tumors and in chemotherapy, hormone therapy, HER2-targeted therapy and anti-immune checkpoint therapy resistance in breast cancer. Furthermore, we discuss the use of TNFα blocking strategies as potential therapies and their clinical relevance for breast cancer. These TNFα blocking agents have long been used in the clinical setting to treat inflammatory and autoimmune diseases. TNFα blockade can be achieved by monoclonal antibodies (such as infliximab, adalimumab, etc.), fusion proteins (etanercept) and dominant negative proteins (INB03). Here we address the different effects of each compound and also analyze the use of potential biomarkers in the selection of patients who would benefit from a combination of TNFα blocking agents with HER2-targeted treatments to prevent or overcome therapy resistance in breast cancer.
... TNF was primarily identified as an anti-tumor factor in the serum of Bacillus Calmette-Guérin-infected mice treated with endotoxin [6,7]. Serum transfer from these mice to other mice with subcutaneously transplanted sarcomas reduced the size of tumors. ...
Background/aims:
Inhibition of tumor necrosis factor (TNF) is an effective treatment for rheumatoid arthritis (RA), but safety concerns about malignancy remain. The aim of this study was to evaluate cancer risk in RA patients treated with TNF inhibitors (TNFi), based on Korean Nationwide Health Insurance claims data.
Methods:
Patients with seropositive RA were selected from the health insurance database containing all citizens' medical information, based on both RA diagnosis codes and medications. Between 2010 and 2014, RA patients treated with conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) and TNFi were enrolled and followed up. We compared the cancer incidence between patients treated with TNFi and csDMARDs using incidence rate ratios (IRRs) after adjustment for age, gender, and observational periods.
Results:
Of 45,423 selected patients with seropositive RA, 2,337 were treated with TNFi and 43,086 were treated with csDMARDs. The TNFi group was younger and was followed-up for a longer duration. During the observational period, 1,732 and 49 cases of cancer were detected in patients treated with csDMARDs and TNFi, respectively. Old age and male sex were associated with cancer occurrence. Adjusted IRRs for all cancers and common cancers demonstrated that cancer incidence did not differ significantly between the TNFi group and csDMARDs group (IRR = 0.913 for all cancers, p = 0.546).
Conclusions:
This study revealed that cancer incidence was similar in RA patients treated with TNFi and csDMARDs. Anti-TNF therapy may be a safe therapeutic option for RA treatment, in terms of malignancy.
... TNF was originally discovered in the serum of animals injected sequentially with a bacterial vaccine and endotoxin. When this serum was administered intravenously, the sarcoma previ- ously transplanted subcutaneously into the mice got necrosed [23,24]. TNF is known as a tumor suppressor, but it also contributes to cancer initiation and tumor progression by cre- ating a permissive microenvironment for tumor invasion and metastasis [25,26]. ...
Transmembrane protein 100 (TMEM100) was first identified as a transcript from the mouse genome. Recent studies have demonstrated that TMEM100 is involved in hepatocellular carcinoma (HCC) malignancy. However, the distribution and clinical significance of TMEM100 in non-small-cell lung carcinoma (NSCLC) remains poorly understood. This study aims to explore the significance of TMEM100 expression in NSCLC. We found that TMEM100 expression was significantly reduced in NSCLC tissues when compared with that in adjacent normal lung tissues (P<0.001). Kaplan-Meier survival analysis showed that overall survival of patients with lower expressions of TMEM100 was significantly shorter (n=152, P<0.05). In addition, TMEM100 overexpression in NSCLC cell lines inhibited cell proliferation in vitro and in vivo. Transwell migration and invasion assay showed that TMEM100 significantly suppressed the migration and invasion of NSCLC cell lines. In contrast, knocking down TMEM100 promoted NSCLC proliferation and migration. Finally, we found that TMEM100 worked as a cancer suppressor gene mainly by inhibiting the TNF signaling pathway. In conclusion, TMEM100 acted as a tumor suppressor in NSCLC and may prove to be a potential prognostic biomarker and therapeutic target for NSCLC.
... Tumor necrosis factor (TNF) α was first identified and characterized according to the ability to induce the regression of tumors in animals [8,9]. TNFα is a pleiotropic cytokine that can affect the growth, differentiation, and metabolism of virtually every nucleated cell type in the body [10]. ...
Synovial mesenchymal stem cells (MSCs) are a candidate cell source for cartilage and meniscus regeneration. If we can proliferate synovial MSCs more effectively, we can expand clinical applications to patients with large cartilage and meniscus lesions. TNFα is a pleiotropic cytokine that can affect the growth and differentiation of cells in the body. The purpose of this study was to examine the effect of TNFα on proliferation, chondrogenesis, and other properties of human synovial MSCs. Passage 1 human synovial MSCs from 2 donors were cultured with 2.5 x 10⁻¹²~10⁻⁷ g/ml, 10 fold dilution series of TNFα for 14 days, then the cell number and colony number was counted. The effect of the optimum dose of TNFα on proliferation was also examined in synovial MSCs from 6 donors. Chondrogenic potential of synovial MSCs pretreated with TNFα was evaluated in 6 donors. The expressions of 12 surface antigens were also examined in 3 donors.2.5 ng/ml and higher concentration of TNFα significantly increased cell number/dish and cell number/colony in both donors. The effect of 25 ng/ml TNFα was confirmed in all 6 donors. There was no significant difference in the weight, or amount of glycosaminoglycan and DNA of the cartilage pellets between the MSCs untreated and MSCs pretreated with 25 ng/ml TNFα. TNFα decreased expression rate of CD 105 and 140b in all 3 donors. TNFα promoted proliferation of synovial MSCs with increase of cell number/ colony. Pretreatment with TNFα did not affect chondrogenesis of synovial MSCs. However, TNFα affected some properties of synovial MSCs.
... A review of the TNF biology (Lebrec et al., 2015) indicates that while TNF was originally described as a necrotic factor in sarcomas (Carswell et al., 1975;Green et al., 1976), blocking the TNF pathway can decrease tumor growth in rodents (Egberts et al., 2008). In addition, TNF affects immune surveillance components both positively and negatively, depending on a broad array of contextual signals, but does not appear to impact anti-Epstein Bar Virus (EBV) immunity, which is often associated with lymphoma in immunocompromised patients (Miceli-Richard et al., 2009). ...
Profound immunosuppression (e.g., AIDS, transplant therapy) is epidemiologically associated with an increased cancer risk, and often with oncogenic viruses. It is currently unclear how broadly this association translates to therapeutics that modulate immunity. A workshop co-sponsored by the FDA and HESI examined how perturbing the immune system may contribute to carcinogenesis, and highlighted priorities for improving non-clinical risk assessment of targeted immunomodulatory therapies. Conclusions from the workshop were as follows. 1) While profound altered immunity can promote tumorigenesis, not all components of the immune system are equally important in defense against or promotion of cancer and a similar cancer risk for all immunomodulatory molecules should not be assumed. 2) Rodent carcinogenicity studies have limitations and are generally not reliable predictors of cancer risk associated with immunosuppression. 3) Cancer risk needs to be evaluated based on mechanism-based weight-of-evidence, including data from immune function tests most relevant to tumor immunosurveillance or promotion. 4) Information from nonclinical experiments, clinical epidemiology and immunomodulatory therapeutics show that immunosurveillance involves a complex network of cells and mediators. To support a weight-of-evidence approach, an increased focus on understanding the quantitative relationship between changes in relevant immune function tests and cancer risk is needed.
... Tumor necrosis factor-alpha (TNF-í µí»¼) is a proinflammatory cytokine with multiple functions in the immune response. Since its initial discovery as a serum factor causing tumor necrosis [17], this potent immune-modulator has also been implicated in a wide spectrum of human diseases including sepsis, diabetes, cancer, collagen tissue diseases, and neurodegenerative diseases181920. In recent years, it has been suggested that TNF-í µí»¼ may participate in apoptotic death of retinal ganglion cells in glaucoma patients [21, 22]. ...
Glaucoma represents a heterogeneous group of optic neuropathies with a complex genetic basis. It is the second-largest cause of blindness in the world that reduces vision without warning and often without symptoms. Among 3 major subtypes of glaucoma, primary open-angle glaucoma (POAG) is the most common form. The focus of this study is to understand the molecular basis of the disease among Indian patients with respect to two genes, Cochlin (COCH) and tumor necrosis factor alpha (TNFA), selected based on reports of possible association with POAG. The genes were screened in patients and controls by PCR and direct sequencing. Although two novel changes (-450 C/T and -79 G/G) were identified in the 5'upstream region of COCH, no causal variant could be identified in either gene. -450 C/T was detected in 3 patients and 2 controls and -79 G/C in a single patient. Further, we did not observe significant association with the promoter SNPs of TNFA that had been previously reported to be associated with POAG pathogenesis. Thus, our study suggests lack of association of both COCH and TNFA with POAG pathogenesis.
Simple Summary
Inflammation has been acknowledged as one of the causes of increased cancer risk. Among the pro-inflammatory mediators, tumor necrosis factor alpha (TNFα) has been identified as an important player in cancer progression and metastasis. On the other hand, TNFα has a central role in promoting innate and adaptive immune responses. These apparently controversial effects are now starting to be uncovered through different studies on TNFɑ isoforms and distinct mechanisms of action of TNFα receptors. The use of immunotherapies for cancer treatment such as monoclonal antibodies against cancer cells or immune checkpoints and adoptive cell therapy, are beginning to broaden our understanding of TNFα’s actions and its potential therapeutic role. This work describes TNFα participation as a source of treatment resistance and its implication in side effects to immunotherapy, as well as its participation in different cancer types, where TNFα can be a suitable target to improve therapy outcome.
Abstract
Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine known to have contradictory roles in oncoimmunology. Indeed, TNFα has a central role in the onset of the immune response, inducing both activation and the effector function of macrophages, dendritic cells, natural killer (NK) cells, and B and T lymphocytes. Within the tumor microenvironment, however, TNFα is one of the main mediators of cancer-related inflammation. It is involved in the recruitment and differentiation of immune suppressor cells, leading to evasion of tumor immune surveillance. These characteristics turn TNFα into an attractive target to overcome therapy resistance and tackle cancer. This review focuses on the diverse molecular mechanisms that place TNFα as a source of resistance to immunotherapy such as monoclonal antibodies against cancer cells or immune checkpoints and adoptive cell therapy. We also expose the benefits of TNFα blocking strategies in combination with immunotherapy to improve the antitumor effect and prevent or treat adverse immune-related effects.
This chapter will, more or less in chronological order, describe work done over a number of years with endotoxin. So many contributions have been made to each topic that to include them all is impossible. The selections here represent works that are considered highly significant since they serve as milestones in the study of this fascinating and challenging substance. Not all important observations can be covered within the limits of this chapter thus, if recognition is not given where it is due, the reader will recognize the difficulties inherent in the author’s task. First we shall look backward on endotoxin research and then try to project the direction it will take in the future.
Endothelial and epithelial cells receive biochemical and biophysical cues from a specialized extracellular environment called the basement membrane. Mimicking the biophysical cues, including the elastic modulus and the structural topography of the basement membrane, is one approach used in tissue engineering to fabricate biomaterials for tissue regeneration. The biophysical properties of many basement membranes present within tissues have been characterized, and the findings highlight that the basement membrane is composed of a compliant protein matrix with nano-fibrous texture. Researchers use various fabrication methods to develop materials that probe how the biophysical scale of the basement membrane control cellular behavior. For example, studies have demonstrated that basement membrane relevant nanotopography can elicit different cell responses (as measured by morphology, proliferation, migration, etc.) compared to micron-scale topography. In addition, cell response to the physical environment is dependant on multiple factors such as the soluble environment and the cell type. These studies demonstrate that biophysical cues are essential components to be included in design of cell studies for tissue engineering.
The cellular parts of the immune system (macrophages, T cells, and B cells) are regulated in their functions, to a large extent, by factors produced by these cells. These factors produced by the cells of the immune system are referred to as cytokines in the generic sense. More specifically, factors produced by lymphocytes are known as lymphokines, whereas those produced by macrophages are known as monokines. Recent progress in biotechnology is based on the production of large amounts of these factors, allowing for clinical evaluation in cancer and AIDS patients as well as other conditions involving the immune system (Figure 3.1).
Metastasis is the leading cause of cancer-related deaths. Cancer stem cells (CSCs) have the distinct ability of self-renewal and proposed capacity to metastasize to distant locations. CSCs are more resistant to radiation and chemotherapeutic agents compared to other cancer cells and, therefore, survive conventional therapies, resulting in local and distant relapse of the disease. This chapter reviews the current knowledge of the metastatic potential of CSCs, termed as metastatic CSCs (MCSCs). Limited evidence has also led to the hypothesis that CSCs may evade host immune responses. This chapter describes the immunosuppressive approaches that cancer cells, including CSCs and MCSCs, employ to evade the host immune system. In addition, the types of immune and inflammatory cells that may be utilized to support tumor progression and promote metastasis are illustrated. Understanding these mechanisms may provide new insights and aid in the development of novel strategies to target the “Achilles’ heel” of CSCs and MCSCs.
To the novice, the immune system presents itself as an extremely complex network of cellular and humoral interactions. This inherent complexity is a reflection of the external world that, by a large variety of attack systems, constantly threatens the integrity of the body. In order to cope with a diverse group of invaders and nonself agents, the immune system had to adapt accordingly and was forced to develop efficient recognition and defense systems (Chap. 1). Among these immunologically specific and nonspecific reactions in both the cellular and humoral compartments, there is a considerable degree of cooperation that requires precise regulation. Beside the well-studied generation of help and suppression in the T and B lymphocyte compartments, it has only recently been fully recognized that non-antibody products of lymphocytes (lymphokines) and macrophages (monokines) may play a prominent role during the induction, maintenance, and effector phases of an immune response. Although lymphokines were first detected in lymphocyte culture supernatants only 20 years ago, this field of immunology has suffered until recently from the lack of a clear biochemical definition of the materials that were generated and employed in a variety of test systems. Usually, a certain lymphokine was named according to a functional response that it generated in a particular in vitro assay. This resulted in a multitude of different names and postulated factors. With the advent of gene technology, it has been possible to improve the characterization of some lymphokines in biochemical terms. This progress has permitted a better study of lymphokine effects in various stages of the immune response.
Within the last half-decade it has become abundantly clear that many of the biological effects of bacterial endotoxin are mediated by what is believed to be a number of different protein and glycoprotein molecules (Berry, 1977; Moore and Berry, 1982). These molecules are derived from cells of the reticuloendothelial system (RES), especially macrophages but from other cells as well. It is intriguing to realize that a diffuse tissue such as the RES qualifies functionally as part of the endocrine system and the mediators as hormones. There is no anatomical or physiological reason why this should not be true since chromaffin tissue, a source of epinephrine and norephinephrine, is also diffusely distributed.
Die Diskussion über Mechanismen der Tumorabwehr möchte ich in den allgemeinen Rahmen der Gast-Wirts-Beziehung stellen. In Analogie zu den Problemen der Infektabwehr, wie sie im Rahmen der medizinischen Mikrobiologie studiert werden, soll formal der autochthon wachsende Tumor als ein dem Wirt wesensfremdes Pathogen betrachtet werden. Dabei sollen heute weniger die krankmachenden Eigenschaften des spontan wachsenden Tumors interessieren. Vielmehr werden im Vordergrund der Diskussion die immunologischen Reaktionsmöglichkeiten stehen, die dem Wirt bei Abwehr eines gegebenen, spontan wachsenden Tumors potentiell zur Verfügung stehen.
The first systematic studies of bacterial vaccines in the treatment of human cancer were performed by W. B. Coley (1891, 1893, 1898, 1906). Additional reports of cancer treatment with such vaccines appeared although not all were of a positive nature (reviewed by Nauts et al., 1946). The use of Coley’s toxins (mixtures of Streptococcus pyogenes and Serratia marcescens) fell into disfavor because of a lack of consistency, which was due, in part, to a lack of potency and a lack of knowledge regarding dosage, site of administration, and frequency of injections. However, a case study by Nauts et al. (1953) revealed the effectiveness of Coley’s toxins when these parameters were taken into consideration. Interest in bacterial products was revived by the experiments of Shwartzman and Michailovsky (1932) and of Duran-Reynals (1935) wherein they showed that bacterial filtrates of gram-negative organisms were effective in producing hemorrhagic necrosis of murine tumors.
Invasive stimuli, including parasitic, bacterial, or viral infection and neoplastic disease, precipitate catabolic changes in cellular metabolism and pathological alterations in the physiology of their mammalian hosts. These disruptions of normal homeostasis, left unresolved, can lead to the depletion of host energy stores advancing to wasting (cachexia), tissue damage, multiple organ system failure, shock, and death. Although such symptomatic patterns are all too familiar to clinicians, the underlying causes of this progressive decline have remained obscure. Because these patterns are frequently attributed to the metabolic demands or direct actions of the pathogen or its products, treatment has quite naturally focused on aggressive elimination of the invading agent and reactive support of failing host physiology. It now appears that invasive stimuli provide a trigger for the release of host-secreted cytokines, and the combined actions of these endogenous mediators elicit most or all of the biologic responses culminating in the pathophysiology of cachexia and shock. This new appreciation for the essential role of host-derived inflammatory mediators has provided a focus for new therapeutic approaches to a broad spectrum of invasive diseases.
In 1891, a surgeon, William Coley, working at The Memorial Hospital in New York City observed the disappearance of a neck tumor in a patient who had contracted erysipelas, a streptococcal infection (1). Thirty eight additional cases dating back to 1868 were also documented by Coley, who then decided to treat other cancer patients with live streptococci. Because this was not “acceptable” medical practice at the time, however, heat-killed organisms were used instead. This approach failed.
As it acts to eradicate viruses, bacteria, and parasitic pathogens, the host immune system may sometimes prove to be a liability rather than an asset. The inflammatory response to invasive organisms may injure the host as surely as the organisms themselves, and, in many instances, the immune response is itself a major pathogenetic vehicle. Thus, physicians strive to temper the immune response in their attempts to manage some infectious disease states. At present, this is achieved through the use of glucocorticosteroids and, occasionally, cytotoxic drugs.
Numerous findings during the past decade have identified an impressive secretory capacity of the phagocytic cells of the reticuloendothelial system (RES). Macrophages in vitro have been found to secrete a variety of potent hormonelike mediators that influence or regulate activities of a variety of other cell types as well as other cellular components of the RES. In many cases, a direct correlation can be made between in vitro findings and established events occurring in vivo during infection or inflammatory responses, reaffirming the importance of the RES in a variety of host-microbial interactions. Of necessity, these mediators have been defined by their in vitro functional activities. As a result, there has been an accumulation of seemingly unrelated data concerning a dramatic number of different mediators. Recent studies, however, indicate that the actual number of different mediators produced by monocytes and macrophages is more limited than that suggested by the available literature. Investigations designed to characterize and to isolate specific mediators have shown that several are in fact the same molecules with multiple functional activities.
An increasing body of data has documented altered cytokine concentrations from Parkinson's disease brains and its rodent models. These changes have been reported in the periphery as well as in the cerebrospinal fluid, where they may be released from a variety of cell types to possibly traffic to and from the brain. Brain resident macrophage, or microglia, is commonly implicated as the cell type contributing to cytokine elevations in the brain during disease. Beyond serving as biomarkers of disease, many studies indicate that the cytokines directly stimulate both neuroprotective and neurotoxic effects on dopaminergic neurons. In particular, rodent models of disease have allowed mechanistic testing of the correlative findings from human diseased brain to suggest that cytokines such as tumor necrosis factor-alpha and interleukin-1β have pleiotropic roles during disease that may vary based upon temporal concentrations and coincident stimulation. © 2014 Springer International Publishing Switzerland. All rights reserved.
The history of fever, studies of its effects and search for its origin, go back as far as the history of medicine. The beneficial effects of fever were praised by famous healers in Egypt, Greece, and in the Roman and Persian empires several millenia ago. This appreciation continued through the Middle Ages into our modern times, when “fever therapy” was applied to diseases as diverse as rheumatoid arthritis (Barsi, 1947), manic depression (Terry, 1939), headaches (Sutherland and Wolf, 1940), neurosyphilis (Favorite and Morgan, 1946), herpes zoster (Bernstein and Klotz, 1947), various eye and skin diseases (Tucker, 1946), and many others.
Endothelial and epithelial cells receive biochemical and biophysical cues from a specialized extracellular environment called the basement membrane (BM). Mimicking the biophysical cues, including the elastic modulus and the structural topography of the BM, is one approach used in tissue engineering to fabricate biomaterials for tissue regeneration. The biophysical properties of many BMs present within tissues have been characterized, and the findings highlight that the BM is composed of a compliant protein matrix with nanofibrous texture. Researchers use various fabrication methods to develop materials that probe how the biophysical scale of the BM controls cellular behavior. For example, studies have demonstrated that BM relevant nanotopography can elicit different cell responses (as measured by morphology, proliferation, migration, etc.) compared to micron-scale topography. In addition, cell response to the physical environment is dependent on multiple factors such as the soluble environment and the cell type. These studies demonstrate that biophysical cues are essential components to be included in the design of cell studies for tissue engineering.
Mononuclear phagocytes, located throughout all tissues of the body, serve a multitude of major defensive and homeostatic functions [1]. One of the most important functions is the pivotal role macrophages can play in host protection against the development and spread of neoplasia [2]. Yet, the consistent and successful immunotherapy of human cancer remains an elusive goal [3]. Understanding the complex mechanisms regulating macrophage-mediated tumor cell destruction may ultimately lead to a rational basis for immunomodulation and, hence, successful immunologically based therapy of neoplastic diseases.
Macrophages kill tumor cells with and without the aid of antibody and evidence suggests that secreted cytotoxic substances are at work in each system. Here Dolph Adams and Carl Nathan discuss the likely involvement in both pathways of several such substances including cytolytic protease and hydrogen peroxide.
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Considerable progress has been made in recent years in our understanding of the cytokines. Three molecules in particular have been the subject of intense research : interleukin-1 (IL-1), interleukin-6 (IL-6) and cachectin (tumor necrosis factor, TNF). They are mainly, although not exclusively, secreted by monocytes and macrophages. Recent results obtained by means of cloning techniques have shown that these cytokines are multi-function proteins which stimulate or modulate numerous biological processes. Although they are biochemically and immunologically distinct, IL-1, IL-6 and TNF frequently show similar activities and interact in various metabolic processes. This general review will deal with the structure of these molecules and that of their respective receptors, together with their metabolism, synthesis and degradation.
The mechanism by which tumour necrosis factors (TNF and lymphotoxin, also called TNFalpha and TNFbeta respectively) exert their cytotoxic activity on many malignant cells, remains largely unknown. Furthermore, the broad array of differentiation (gene induction) and mitogenic activities towards many primary cells is still a subject of intensive investigation. TNF is an important mediator in inflammation, immune responses and infection-related phenomena and these activities contribute to the severe toxicity seen when TNF is used as an anticancer agent. The first step in the mechanism of action is the specific binding of the ligand to its receptors and dissection of the molecular mechanism involved in this interaction is the subject of this review. The reasons for the interest in this aspect are obvious: first, the development of strong antagonistic TNF analogues can be useful in dampening the potentially lethal or debilitating effects of an overproduction of the cytokine (as in septic shock or rheumatoid arthritis). Secondly, since two distinct TNF receptors exist, construction of TNF muteins that distinguish between both types may lead to derivatives of this pleiotropic agent with a more restricted biological activity pattern. Ideally, one would like to develop a TNF mutant that has retained its cytotoxic action on tumour cells without inducing the deleterious systemic toxicity. Such an optimized TNF molecule could become a potent anticancer agent.
Morphologic and metabolic studies have been carried out on Friend leukemia cell (FLC) tumors (grown in DBA/2 mice) or fibrosarcomas (grown in C3H/HeN or C3H/Hej mice) shortly after peritumoral injection of recombinant mouse tumor necrosis factor (TNF) α. Marked vascular congestion and focal extravasation of erythrocytes were observed as soon as I hr after injection of either FLC tumors or fibrosarcomas with TNF. Focal areas of disaggregation of tumor cells were observed I hr after injection of TNF. Intraluminal thrombi (composed of degranulated platelets and fibrin) were detected 3 and 6 hr after TNF treatment, and were associated with areas of depletion of endothelial cell cytoplasm. To correlate these morphologic changes in the tumor with alterations in tumor metabolism, NMR spectroscopy and biochemical studies were undertaken on freshly dissected FLC tumors and fibrosarcomas shortly after injection of TNF. The earliest metabolic alterations observed after I hr in TNF-treated FLC tumors or fibrosarcomas were: (i) increase in the average intratumoral PH; (ii) decrease in the levels of ATP. These phenomena were not associated with a reduced glycolytic capacity of TNF-treated tumors as, at these early times after injection, the levels of lactic acid were virtually the same for TNF-treated or control treated tumors. Alterations in the levels of some products of phospholipid degradation (Gro-PCho, GroPEtn, Crop and Cho) also occurred in these tumors as early as 3 hr after TNF treatment. These metabolic changes were not observed in ascitic FLC tumors after TNF treatment. We suggest that TNF induces alterations in tumor blood vessels which subsequently lead to changes in tumor metabolism and tumor degeneration.
: Polyclonal antibodies to ganglioside Gmi have been prepared and characterised by direct and competitive enzyme-linked immunoassay. An immunoglobulin fraction was prepared from a rabbit antisera showing high specificity and antibody titre for GMI relative to the other major brain gangliosides. The anti-GMI immunoglobulin fraction and B-cholera toxin specifically labelled neurons in primary cultures of embryonic chick dorsal root ganglia and there was a good correlation between the relative increase in binding of anti-GMI immunoglobulin and B-cholera toxin following neuraminidase treatment of a variety of cell types. At antibody concentrations that show saturable binding to endogenous ganglioside in the neuronal membrane, the anti-GM1 immunoglobulin fraction did not interfere with the nerve growth factor (NGF)-mediated fibre outgrowth and neuronal survival as indexed by measurement of neu-rofilament protein levels. Similarly, at levels in excess of those shown to stimulate thymocyte proliferation, B-cholera toxin was also without effect. These data are not consistent with GMI in the neuronal membrane functioning as a receptor molecule for NGF and/or other differentiation factors present in the tissue culture media.
Tumor necrosis factor (TNF) was originally described as a circulating factor that can induce hemorrhagic necrosis of tumors. It is now clear that TNF has many different functions in cancer biology. In addition to causing death of cancer cells, TNF can activate cancer cell survival and proliferation pathways, trigger inflammatory cell infiltration of tumors, and promote angiogenesis, and tumor cell migration and invasion. These effects can be explained by the diverse cellular responses TNF can initiate through distinct signal transduction pathways, opening the way for more selective targeting of TNF signaling in cancer therapy.
TNF-α is a highly versatile cytokine, playing an important role in both apoptosis and inflammation, which is central to reproduction. It belongs to the Th1 type of cytokines. Thus, TNF-α is a proinflammatory cytokine and is involved during follicle development and ovulation, corpus luteum formation and regression and cyclic endometrium function. It has been postulated to be detrimental to the survival of the conceptus due to apoptosis of human primary villous trophoblast cells, leading to miscarriage. However, TNF-α may have a dual role in early pregnancy. Hormonally regulated uterine TNF-α in the mother is probably essential for trophoblast cell invasion, while increased production of TNF-α by maternal macrophages may facilitate labor. The current knowledge on the role of TNF-α and its receptors in the female reproduction is described in this review.
Abstract In the days before effective anti-inflammatory drugs were available, the treatment of intraocular inflammation was very difficult. A popular treatment before 1950 was nonspecific protein shock therapy. Boiled milk and typhoid vaccine were the principal proteins used. In some way a fever was produced, followed by changes in the blood picture referred to as a febrile hemogram. Because the rise in temperature was the most obvious and easily recorded manifestation, it was the best criterion of the severity of the reaction. Pitfalls, mechanism of action, techniques of administration, and contraindications of this method will be discussed.
A distinctive glycopeptide, which acts as an acceptor for a cancer-associated galactosyltransferase, has been detected in sera and effusions of patients with extensive carcinoma. Cancer-associated galactosyltransferase acceptor (CAGA) purified from human malignant effusions was tested for its effects on cell growth in vitro and in vivo. Addition of the glycopeptide to the media of cells growing in tissue culture significantly inhibited the attachment and growth of transformed cells but had minimal effect on nontransformed cells. Transformed hamster cells (BHKpy, BHKpygiv, Nilpy) and human malignant cells (BT-20 human breast, pancreatic and colonic carcinoma cells) were killed by the addition of as little as 0.5 μg of acceptor (per ml of medium); whereas nontransformed counterparts did not show a significant change in growth or morphology. In vivo studies showed that the acceptor inhibited development and progression of tumors in hamsters inoculated with tumorigenic BHKpy cells and in nude mice inoculated with human carcinoma cells. Growth of tumors was inhibited 69–94% in animals given 20 μg of acceptor subcutaneously and 39–67% when acceptor was given intraperitoneally at the time of tumor cell inoculation. Administration of the acceptor after the development of palpable tumor (±0.5 cm) caused a 60–85% reduction in growth rate and, in some cases, actual reduction in size and disappearance of palpable tumor. These studies demonstrate that a galactosyltransferase glycopeptide acceptor purified from human malignant effusions produces selective inhibition of transformed cell growth in animal and tissue culture systems.
In this study a variety of immunostimulatory agents was tested alone or in combination with other agents to measure their ability to enhance nonspecific resistance to challenge with the TA3-Ha transplantable murine ascites tumor. Lipopolysaccharides (LPS) were effective alone in protecting mice from tumor. Their lipid-free, nontoxic, polysaccharide-rich hydrolytic products (PS) showed a much reduced, but still significant effect in anti-TA3-Ha resistance induction. Other agents that stimulated tumor resistance included nontoxic native hapten and trehalose-6,6-dimycolate (P3) of Ribi, synthetic glycolipids, cord factor, and killed BCG preparations. Simultaneous pretreatment with a combination of any of these agents and either LPS or PS resulted in a significantly higher level of tumor resistance. Administration of LPS, PS, or native hapten to mice that had been previously infected with viable BCG resulted in the strongest antitumor effect. These studies demonstrate that nonspecific resistance can be enhanced in an additive or synergistic manner by using combinations of agents, which presumably stimulate different cell populations of the host's immune system.
Endotoxin induced necrosis of the Meth A mouse tumour has been investigated using macroscopic, histological and ultrastructural examination methods.On the 8th day after tumour cell transplantation, the animals received a relatively non-toxic dose of the Salmonella abortus equi endotoxin intravenously. The natural history of the tumour necrosis took the following course:1.
The earliest morphological changes could be seen with the electron microscope 90 min after administration of the endotoxin, and were seen as an interstitial oedema with separation of the tumour cells.
2.
Haemmorrhagic necrosis of the tumour was complete 4 hours after injection, and could be easily recognized with the naked eye.
3.
Rejection of the necrotic malignant tumour was complete two weeks after LPS administration. Only minor residual scarring of the belly-wall remained.
Haemorrhagic tumour necrosis due to endotoxin can be compared with the localized Shwartzman reaction and probably involves tumour necrotizing factor (TNF). For complete destruction of a tumour by haemorrhagic necrosis the size of the tumour is critical. Certain regression after endotoxin administration depends upon additional T-cell-mediated immunity (provided the tumour is immunogenic).In contrast to the haemorrhagic necrosis, BCG-induced tumour regression is accompanied by granulomatous inflammation, which may be responsible for destruction of the tumour.
Soluble tumor necrosis factor receptor (sTNF-R) is known to inhibit patient immunity via specific binding with the TNF molecule. To examine the possible involvement of sTNF-R in cancer immunotherapy, the plasma levels of sTNF-R of both 55 kDa and 75 kDa origins were estimated when TNF was induced in patients with malignancy using both a polysaccharide preparation (Lentinan) and a streptococcal preparation (OK-432). The pretreatment plasma levels of the 55kDa and 75 kDa sTNF-R were 1.04 0.53 and 1.06 0.34 ng/ml (mean SE), respectively. The plasma levels of TNF were undetectable before treatment. The plasma sTNF-R levels peaked 2 h after the administration of OK-432 and followed the same pattern as the TNF levels in plasma. Both TNF and sTNF-R nearly returned to pretreatment levels at 16h after the induction of TNF. The peak plasma levels of the 55kDa and 75 kDa sTNF-R were 2.46 0.95 and 3.03 0.88 ng/ml, respectively, but they did not correlate with the plasma TNF levels. When peripheral white blood cells were cultured with the addition of lipopolysaccharide in vitro, an elevation of the 72 kDa sTNF-R was detected. Thus, the plasma source of this soluble receptor can at least be partly attributed to the white blood cells. However, the 55 kDa sTNF-R showed little increase in the cultures, and its source remains unknown. We should therefore be aware of the elevation of plasma sTNF-R levels by the induction therapy of TNF for patients with malignancies because of the immunosuppressive effect of sTNF-R.
The endogenous induction of tumor necrosis factor serum (TNS) for cancer immunotherapy was undertaken in the immediate postoperative
period using Lentinan as the primer and OK-432 as the inducer. The changes in several immunological markers of the blood were
assayed and compared with a control group to clarify the effects of this treatment. Plasma TNF-α levels were elevated two
to three hours after eliciting treatment. The neutrophil count was elevated on the 7th post-operative day (POD) and the natural
killer (NK) cell activity was transiently suppressed on the 1st POD, but NK cells possessing a high activity (Leu7-CD16+)
were preserved until the 7th POD. Helper/inducer (CD4+) and killer cells (CD8+CD11−) tended to increase, and suppressor (CD8
bright+ CD11+) cells tended to decrease in the induction group. There was no difference in the levels of prostaglandin E2 (PGE2) between the groups, but a marked elevation of interferon-γ was evident on the 1st POD in the induction group. This treatment
may be useful as postoperative adjuvant immunotherapy for cancer due to its ability to induce cytokines and activate host
immune mechanisms.
The thermolabile α-galactosidase (α-galactosidase A) and thermostable α-galactosidase (α-galactosidase B) were separated and purified from human placenta. A homogeneous α-galactosidase B preparation was obtained, but the α-galactosidase A preparation contained small amounts of contaminating protein and various other acid hydrolase activities. Each preparation had a molecular weight of approximately 150,000, as estimated by Sephadex filtration. α-Galactosidase A had a Km of 3.4 mm for the artificial substrate, 4-methylumbelliferyl-α-d-galactopyranoside, and of 40.6 mm for melibiose. α-Galactosidase B hydrolyzed 4-methylumbelliferyl-α-d-galactopyranoside with first order kinetics and appeared to have no activity with melibiose. Both enzymes had maximal enzyme activity at pH 4.5, but α-galactosidase A had a broad pH-activity curve, while that of the B enzyme was sharply peaked. α-Galactosidase A was inhibited by myoinositol; α-galactosidase B was not. The isoelectric point of α-galactosidase A was 4.70 ± 0.07; the isoelectric point of α-galactosidase B was 4.42 ± 0.04.
Antibodies were produced against both the α-galactosidase A and α-galactosidase B preparations. No cross reactivity between the two enzyme preparations was found on double immunodiffusion. Neither antiserum neutralized enzyme activity, but the anti-α-galactosidase A serum precipitated α-galactosidase A activity from solution and the anti-α-galactosidase B serum precipitated α-galactosidase B activity from solution. Treatment of α-galactosidase A with neuraminidase does not change its immune reactivity or kinetic properties.
These studies lend no support to the concept that α-galactosidase A is the neuraminyl derivative of galactosidase B or that the two enzymes are closely structurally related.
Double stranded RNA (dsRNA) whether isolated from a fungal virus or prepared synthetically (i.e., Poly I Poly C) and endotoxin were found to exert very similar effects on syngeneic murine lymphomata and fibrosarcomata. They cause complete regressions of some established subcutaneous (s.c.) or intradermal (i.d.) tumours but not of intraperitoneal (i.p.) tumours when administered either systemically or directly into the tumour. To achieve this effect the tumours must be fully established and the best results were obtained when treatment was started 7 days after transplant. If treatment is started within the first 3 days following the transplantation of the tumour then only a slight inhibition of growth rate was observed. These agents can also act prophylactically and protect mice against a subsequent challenge but only if this is given i.p. and not if given s.c. or i.d. The prophylactic action is explained by the action of dsRNA and endotoxin on peritoneal macrophages which cause them to become cytotoxic to tumour cells (i.e., to become activated).
The therapeutic effect of systemically administered endotoxin and dsRNA on established tumours is not the result of a direct action on the tumour cells themselves but is a complex process requiring the co-operation of several host factors. Haemorrhagic necrosis involving coagulation is essential (i.e., heparinization reduces the regression of tumours) but is not itself sufficient. Immunosuppression by whole body irradiation or by antilymphocyte serum also interferes with the antitumour action of dsRNA and endotoxin in spite of the fact that haemorrhagic necrosis still occurs. Also, the magnitude of the antitumour action correlated in a series of different tumours with their antigenicity. The observed tumour regressions are probably brought about by (1) vascular damage in the tumour which permits immune defence mechanisms of the host to gain access to the tumour and (2) activation of macrophages present within the tumour. The relative contribution of these two mechanisms may depend on the nature of the tumour and the route of administration of the active agents.
Dibenyline, which protects against the lethal action of endotoxin by preventing the action of the catecholamines on the α-adrenergic receptors, makes it possible to increase the effectiveness of endotoxin in tumours by allowing a large dose to be given. Lipid A, a derivative of endotoxin which does not contain polysaccharide, has similar antitumour action to dsRNA and endotoxin. Some common features of the chemical structure of lipid A and dsRNA are discussed.
Evidence is presented suggesting that the apparent non-specificity of pyrogenic tolerance observed with Gram-negative bacterial endotoxins is due to related antigenic determinants associated with the macromolecular toxins. This is based on results obtained in rabbits from pyrogenic cross-tolerance tests with selected endotoxins. In these tests, purified endotoxins from Escherichia coli (COO8) and Chromobacterium violaceum (CV) gave results one might expect with non-reciprocal cross-reacting antigens in classical immune systems. Additional evidence for an immune mechanism in tolerance is suggested by the highly significant anamnestic response observed.
Lipid A, a toxic derivative of the purified COO8 endotoxin, failed to induce pyrogenic tolerance against the parent toxin. These results are explained by assuming that endotoxins have two interdependent activities associated with different portions of the macromolecule; one is assumed to be responsible for the primary toxicity, and the other is involved in secondary toxicity. The latter is dependent on the hypersensitive state of the host. Additional evidence for the role of hypersensitivity in secondary toxicity is based on the observation that adult rabbits are highly sensitive to the pyrogenic, lethal, and skin-reacting activities of endotoxin in contrast to young animals which are more resistant to all of these attributes of toxicity.
In adults, the host responses to pyrogenicity, lethality, and skin reactivity could be partially inhibited by the early exposure of the animals to massive doses of endotoxin equivalent to a LD50. The pyrogenic tolerance shown in these animals was specific indicating that the inhibition of the hypersusceptibility to endotoxin involved an immunological mechanism.
A mechanism of endotoxin tolerance is proposed and discussed based on the induction of specific antibodies capable of assisting the RES in the clearance and destruction of endotoxin.
It is suggested that the present inconsistencies relative to the chemical nature and biological activities of endotoxins might be explained on the basis of these two activities and the failure to recognize the importance of the immunological state of the host in which the toxins are tested.
1. 1. A rapid and reproducible method for measurement of lysozyme activity is described. 2. 2. Using this method, the ultra-violet inactivation of lysozyme has been studied and found to conform to a first order reaction. 3. 3. The measured quantum yield for inactivation at 2537 A is 0.024 and remains unaltered over the pH range 3.6 to 12.0. A small effect of concentration on quantum yield is indicated. 4. 4. Inactivation is accompanied by photo-oxidation and other secondary processes, with no apparent liberation of free amino acids or peptides. 5. 5. The relation of molecular weight to quantum yield, as well as the other findings, are discussed.
In studying "hemorrhagic necrosis" of tumors produced by endotoxin, it was found that the serum of bacillus Calmette--Guerin (BCG)-infected mice treated with endotoxin contains a substance (tumor necrosis factor; TNF) which mimics the tumor necrotic action of endotoxin itself. TNF-positive serum is as effective as endotoxin itself in causing necrosis of the sarcoma Meth A and other transplanted tumors. A variety of tests indicate that TNF is not residual endotoxin, but a factor released from host cells, probably macrophages, by endotoxin. Corynebacteria and Zymosan, which like BCG induce hyperplasia of the reticulo-endothelial system, can substitute for BCG in priming mice for release of TNF by endotoxin. TNF is toxic in vitro for two neoplastic cell lines; it is not toxic for mouse embryo cultures. We propose that TNF mediates endotoxin-induced tumor necrosis, and that it may be responsible for the suppression of transformed cells by activated macrophages.
The cytotoxic activity of activated macrophages against tumorigenic target cells appears to be mediated by lysosomal enzymes of activated macrophage origin. Lysosomes of activated macrophages are secreted directly into the cytoplasm of susceptible target cells, which subsequently undergo heterolysis. This reaction can be inhibited by agents which prevent the exocytosis of macrophage lysosomes (hydrocortisone) or which interfere with the action of lysosomal enzymes (trypan blue).
The endotoxically active cell wall glycolipid of a highly deficient heptose-less R mutant of Salmonella minnesota, consisting of 2-keto-3-deoxyoctonic acid (KDO) and lipid A, was subjected to hydrazinolysis. Besides fatty acid hydrazides, a water-soluble main fraction was obtained, containing KDO, glucosamine, and ester phosphate and free phosphorylethanolamine. The application of two procedures of partial hydrolysis, followed by paper electrophoretic separation, led to seven oligosaccharides.
Their structures were evaluated with the aid of the following analytical methods: further acid degradation, borohydride reduction, ninhydrin degradation, the Morgan-Elson reaction, treatment with phosphomonoesterase and N-acetylglucosaminidases. The results show that the backbone of lipid A contains phosphorylated glucosaminyl→glucosamine disaccacharide units, probably linked β-1,6, to which a trisaccharide composed of three KDO residues is linked ketosidically to form a pentasaccharide which appears to be a common structure in many Enterobacteriaceae.
The cytotoxicities of double stranded RNA and endotoxin have striking similarities. Both seem to render mouse macrophages cytotoxic to other mouse cells.
Gelation of a lysate of amebocytes of Limulus, the horseshoe crab, is a sensitive indicator of the presence of a variety of endotoxins. Amebocyte lysate is stable. The reaction between lysate and endotoxin is reproducible and easy to measure. The rate of the reaction between amebocyte lysate and endotoxin is dependent upon the concentration of endotoxin. The reaction is capable of detecting as little as 0.0005 μg per milliliter of endotoxin in human blood plasma and serum. Addition of endotoxin to normal blood resulted in loss of detectable endotoxin activity when undiluted plasma or serum prepared from such blood samples was compared with saline controls. Coagulation resulted in additional loss of endotoxin activity. Preincubation of endotoxin with blood or a low calcium ion concentration was not necessary for the demonstration of inhibition. The inhibitory effect could be eliminated almost completely by dilution of serum or plasma to 1 to 10 per cent. Extraction of plasma with chloroform for 60 minutes permitted essentially total recovery of endotoxin which had been added to whole blood or plasma. The increase in positive tests, following dilution or chloroform extraction of serum or plasma, suggested that the inhibitory nature of blood is primarily the result of reversible binding between endotoxin and serum protein(s), rather than destruction of endotoxin. This test is the most sensitive in vitro assay for endotoxin which has been reported. The technique should have application both for clinical and experimental studies of endotoxin and endotoxemia.
A new and simplified procedure for the automated determination of serum alkaline phosphatase uses the AutoAnalyzer. The substrate p-nitrophenylphosphate in 2- amino-2-methyl-l-propanol offers the prime advantage of providing directly its own chromogen, p-nitrophenol, following enzyme action. The procedure also permits use of small samplevolumes,provideslinear reaction rates, and has a simple manifold de- sign. Correlations are presented between the manual procedure and the automated system. T1HIS PAPER reports the development of a simplified automated proce- dure for the determination of serum alkaline phosphatase. The method, developed for use with the AutoAnalyzer,t utilizes a p-nitrophenyl-
A procedure is presented for the automateddetermination of acidphosphatase activity in biologicalmaterialsfrom either plant or animalorigin. The TechniconN-i flow diagramhasbeensimplified,and the reagentsfor the measurement of enzymatically producedphenolicsubstances havebeenmodifiedwithout lossof rangeor sensitivity. Bothphenyiphosphate anda-naphthylphosphate, introducedby Babson et a!. (2, 3) for serumacid phosphatase, maybe usedas substrates.The Emersonreaction (alkaline aminoantipyrine and ferricyanide)with a-naphtholformsa more stableand repro- duciblecolor than the coupledproduct with tetrazotized o-dianisidine(Babson). Supporting data for thesemodifications are included. 1'JHIs 1'APER I)Iesemlts a siniplifled procedlure for tile automated deter- mination of acid phosphatase, which is apphcabie to material of either plant or animal origin. The modified flow diagram and reagents for both the substrate amid tile coiorimetric reactions can be used interchangeably for the expression of enzyme activity in terms of either phenol (phenyl- phosphate substrate) or -naphthol (-naphthylphosphate substrate) equivalents.
1. Correlation between elution volume, V(e), and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 (alpha-crystallin) on Sephadex G-200 columns at pH7.5. 2. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free globular proteins fitted a smooth V(e)-log(mol.wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. 3. V(e)-log(mol.wt.) curves based on results with the three gels are taken to represent the behaviour of ;typical' globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. 4. Some glycoproteins, including gamma-globulins and fibrinogen, do not conform to the standard relationship. The effect of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. 5. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. 6. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually >10(6). 7. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250000. The free sub-units readily lose enzymic activity. 8. Recognition of the atypical gel-filtration behaviour of gamma-globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140000; milk alkaline phosphatase, 180000.
This chapter discusses the different aspects of thiobarbituric acid assay of sialic acid. Periodate oxidation of the neuraminic acid backbone of sialic acids results in the formation of β-formylpyruvic acid from carbon atoms 1 to 4. The N-acetyl or N-glycolyl group of sialic acids apparently does not interfere with periodate oxidation. β-Formylpyruvic acid is coupled with 2-thiobarbituric acid to form a red chromophore with a maximum absorption at 549 mμ. It is found that as only free sialic acids are reactive in the assay, hydrolysis of sialic acid-containing material must be carried out for the measurement of total sialic acids. The assay is suitable for measuring the release of bound sialic acid by sialidase. A series of 2-keto, 3-deoxy sugar acids, found in bacteria, also react in the thiobarbituric acid assay. These produce a chromogen with a peak at 549 mμ. They can be readily distinguished from sialic acids because they are not reactive in the orcinol or direct Ehrlich assays for sialic acids. The hydrolysis frees all the sialic acids from several mucoproteins that have been tested except for brain tissue where release of sialic acids takes place for several hours.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
- L Warren
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