Potent Inhibition of Interleukin 1β-Mediated Human Melanoma (A375.6) Lysis by Corticosteroids,Staurosporine, and Tilorone

Abstract

The mechanism of human interleukin (IL)-1 β-mediated cytolysis was studied in a human melanoma cell line, A375.6. Purified recombinant human IL-1β produced 50% cytocidal activity at 50 pg/ml. A variety of compounds were tested for their ability to interfere with A375.6 lysis. Compounds were added simultaneously with IL-1β (100 pg/ml), and tumor cytolysis was measured after 72 hr of culture by release of ¹²⁵I from DNA of A375.6 cells labeled with [¹²⁵I]-dUrd. A variety of antiinflammatory/immunosuppressive agents (including auranofin, chloroquine, cyclosporin A, d-penicillamine) and several cyclo-oxygenase/lipoxygenase inhibitors (AA-861, BW755c, and indomethacin) lacked protective activity. Similarly, phospholipase inhibitors (mepacrine and 4-bromophenacyl bromide), putrescine, inhibitors of lysosomal activity (chloroquine and NH4CI), calcium channel blockers (nifedipine and verapamil), calmodulin inhibitors (W-7 and calmidazolium), and inhibitors of ADP ribosylation (nicotinamide and 3-aminobenzamide) were inactive. In contrast, corticosteroids (dexamethasone, hydrocortisone, and paramethasone acetate), tilorone, and protein kinase C inhibitors (1-[5-isoquinolinyl-sulfonyl]-2-methylpiperazine and stauro-sporine) significantly inhibited IL-1 β-mediated A375.6 cytolysis. These compounds also interfered with tumor necrosis factor-mediated lysis of A375.6, suggesting common mechanisms of tumor cytotoxicity by these monokines. This model may be useful for delineating intracellular biochemical events integral to IL-1 action.     

To what extent are free testosterone (FT) values reproducible between the two Washingtons, and can calculated FT be used in lieu of expensive direct measurements?

Free testosterone (FT) measurement by equilibrium dialysis and liquid chromatography-tandem mass spectroscopy (LCMS/MS) is the "gold standard." We hypothesized that calculated FT values could substitute for measured values; compared FT results reported by Walter Reed Army Medical Center (WRAMC), Washington, DC, with results reported by the Seattle Veterans Affairs Health Care System, Seattle, WA, for 3 patient groups; and evaluated the calculated FT values by gold-standard measurements. Groups 1 and 2 included samples from 54 patients evaluated in Seattle and 94 evaluated at a primary care clinic in Alaska whose samples were analyzed in Seattle, respectively, whose care resulted in ordering an FT measurement. Group 3 included samples from 64 patients evaluated in endocrine WRAMC clinics. Calculated FT values between the 2 facilities demonstrated a strong correlation (R2 = 0.98) for all 212 patients. In a comparison of calculated FT values with measured levels, group 3 had an R2 = 0.93; however, samples with FT values less than 50 pg/mL had a poorer correlation (R2 = 0.45). Calculated FT values may accurately reflect and be substituted in the clinical setting for gold-standard values when levels are more than 50 pg/mL.     

Adhesion of Human Myelomonocytic (HL-60) Cells Induced by 1, 25-dihydroxyvitamin D3 and Phorbol Myristate Acetate is Dependent on Osteopontin Synthesis and the αvβ3 Integrin

A key event in bone resorption is the attachment of osteoclasts on the bone surface. Accumulating data supports the notion that this interaction involves the matrix protein osteopontin on the bone surface interacting with a receptor of the integrin family (cx_vP₃) at the osteoclast clear zone. Based on the recent observation that osteopontin phosphorylation appears to be required for this interaction, it is of considerable interest to delineate the structural requirements for osteopontin-mediated cell attachment. Although binding of isolated osteoclasts to osteopontin-coated surfaces involves the Ovp₃ integrin, this system suffers from considerable disadvantages to allow detailed studies in this respect. We have therefore turned to another cell system, HL-60 promyelocyte leukemia cells, to address these questions. In the presence of the phorbol ester TPA (10 nM) and 1, 25-dihydroxyvitamin D₃ (100 nM), 90% of the HL-60 cells became adherent within 24 hours and exhibited a macrophage-like appearance. Under these conditions, the osteopontin mRNA levels was elevated around 60-fold compared to the control, non-adherent cells. The absolute requirement of de novo osteopontin synthesis for the TPA and 1, 25-vit 1), -induced HL-60 cell adhesion was demonstrated by neutralisation of adhesion using an anti-osteopontin polyclonal antibody as well as following transfection of an anti-sense osteopontin phosphorothioate-modified oligonucleotide. Finally, inhibition of induced HL-60 cell adhesion by an RGD-containing peptide or by an antibody to the α_vβ₃ integrin complex suggested that the cell-derived osteopontin interacts with this integrin. It is concluded that HL-60 cells induced to differentiate with the combination of TPA and 1, 25-vit D₃ can be utilised as a model system to delineate structural requirements involved in the interaction between osteopontin and the αvβ₃ integrin.     

The Changes in the T helper 1 (Th1) and T helper 2 (Th2) Cytokine Balance During HIV-1 Infection are Indicative of an Allergic Response to Viral Proteins that may be Reversed by Th2 Cytokine Inhibitors and Immune Response Modifiers – a Review and Hypothesis

The HIV-1 infection in humans induces an early cellular immune response to react to the viral proteins with a cytotoxic T cell (CTL) response that fails to inhibit virus replication and the spread of the virus. It became evident that the progression of the disease causes chronic changes to the immune system of which a gradual increase in IgE antibodies is one of its features. When the HIV-1 epidemic began, the relation between the gradual increase in IgE content and AIDS was not understood, but later it became a marker for disease prognosis. The advances in the knowledge on T helper 1 (Th1) and T helper 2 (Th2) cells revealed that Th1 cells produce cytokines that stimulate the proliferation of CTLs. Th2 cells produce cytokines that are responsible for the activation of the humoral immune response in healthy people. Studies on both Th1 and Th2 cytokine synthesis revealed an aberration in HIV-1 infected people. Clerici and Shearer presented a hypothesis (1993) whereby Th1 cell activity declines and Th2 activity increases (the Th1 --> Th2 switch hypothesis) in HIV-1 infected people. In fact, experiments concerning this hypothesis ultimately supported the premise that the switch involves a critical change in the cytokine balance, which leads to the contraction of AIDS. However, the research community must still discern why such a Th1 --> Th2 switch takes place in infected people and how it can be reversed. The present review points to the fact that a similar Th1 --> Th2 switch constitutes the response of allergic people to environmental allergens. HIV-1 patients and allergic people that are exposed to allergens respond with an increased synthesis of Th2 cytokines and IgE, together with a decrease in Th1 cytokines. The studies on allergen-induced Th2 cells revealed that the Th2 cytokine IL-4 induces B cells to synthesize IgE, and cytokine IL-5 is the inducer of eosinophilia, just as in HIV-1 infection. The difference between the HIV-1 infection and allergies is the ability of IL-4 to induce the synthesis in T cells of the HIV-1 coreceptor CXCR4 that selects from the replicating virus a syncytium-inducing (SI) virus, a variant virus that replicates rapidly. The present hypothesis implicates the viral proteins in the induction of Th2 cytokine synthesis. This suggests that in viral proteins, allergen-like domains may be responsible for the activation of Th2 cytokine synthesis. Based on the analogy of the responses of humans to allergens and HIV-1, the following hypotheses is suggested: (a) Removal of allergen-like domains from viral genes by genetic engineering may provide viral proteins for vaccine development. (b) Attempts to treat allergic patients with IL-4 receptor inhibitors suggests that the "Th2 --> Th1 Reversion" constitutes a possible approach to inhibiting the Th2 cytokines and inducing a revival of the anti-viral Th1 response.     

Respiratory syncytial virus (RSV) evades the human adaptive immune system by skewing the Th1/Th2 cytokine balance toward increased levels of Th2 cytokines and IgE, markers of allergy—a review

Infection of infants in their first year of life, children and elderly people with the respiratory syncytial virus (RSV) endangers the life of the patient. An attempt to develop a formalin-inactivated RSV (FI-RSV) vaccine during the 1960s resulted in an aggravated infection in immunized children, leading to hospitalization, while infection of non-immunized children produced much milder symptoms. The reason for this remained an enigma, one which was gradually solved over the last decade by many researchers who studied the molecular biology of RSV infection of respiratory ciliary cells. Clinical studies of RSV-infected patients indicated increased levels of Th2 cytokines and IgE in the patients’ sera, suggesting that an allergy-like condition developed during infection. The biomarkers of allergy caused by endogenous or environmental allergens include a marked increase of the Th2 cytokine IL-4 and IgE non-neutralizing antibodies to the allergen. The way allergens trigger allergy was deciphered recently, and will be discussed later. Studies of RSV infection led to the suggestion that RSV patients suffer from allergy prior to RSV infection, a concept that was later abandoned. Studies on HIV-1 [Y. Becker, Virus Genes 28, 319–331 (2005)] research led me to the hypothesis that since HIV-1 infection induces a marked increase of IL-4 and IgE in serum, an allergy-like condition, the AIDS stage is the result of an allergen motif that is embedded in the shed viral gp120 molecules. It is hypothesized that the viral-soluble G glycoprotein (sG) contains a T cell superantigen (Tsag) that is capable of binding to the V_H3 domain of IgE/FcεRI⁺ hematopoietic cells, basophils, mast cells and monocytes, similar to the case of allergens, and that this aggregation causes these innate system cells to degranulate and release large amounts of Th2 cytokines (IL-4, IL-5, IL-10, IL-13) into the blood. The way these Th2 cytokines skew the Th1/Th2 balance toward Th2 > Th1 will be discussed. The aim of the present review is to base RSV pathogenicity on the numerous very good analyses of the virus genes and to suggest a therapeutic approach to treatment that is directed at preventing the inhibitory effects of Th2 cytokines on the adaptive immune system of the patients, instead of inhibiting RSV replication by antivirals. The review of the molecular research on the role of the viral fusion (F) and attachment (G) glycoproteins of RSV provided information on their role in the virus infection: early in infection the F glycoprotein induces Th1 cells to release the Th1 cytokines IL-2, IL-12 and IFN-γ to activate precursors CTLs (pCTLs) to become anti-RSV CTLs. The G and sG glycoproteins attach to FKNR1⁺ ciliary respiratory epithelial cells as well as directly to eosinophils to the lungs. The sG T cell antigen can also induce the release of large amounts of Th2 cytokines from CD4⁺ T cells and from FCεRI⁺ mast cells, basophils and monocytes. By comparison to HIV-1 gp120 it is possible to show that in the G and sG proteins the T cell antigen resembles the CD4⁺ T cell superantigen (=allergen) domain of HIV-1 gp120 which aggregates with IgE/FCεRI⁺ hematopoietic cells. The increased IL-4 level in the serum inhibits the adaptive immune response: IL-4Rα⁺ Th1 cells stop Th1 cytokine synthesis and IL-4Rα⁺ B cells stop the synthesis of antiviral IgG and IgA and switch to IgE synthesis. In addition, the hematopoietic cells release histamine and prostaglandin which induce wheezing. The gradual increase of sG molecules creates a gradient of fractalkine (FKN) which directs IL-5-activated eosinophils to the lungs of the patient.     

Comments on the article “Changing attitudes towards the care of children in hospital: a new assessment of the influence of the work of Bowlby and Robertson in the UK, 1940–1970” by Frank C.P. van der Horst and Rene van der Veer (Attachment and Human Development Vol 11, No 2, March 2009, 119–142)

The authors give an impressive list of references, but these do not reflect the situation in the UK; most of those looking after children in hospital did not write about what they did or read about what others did. Children in hospital saw little or nothing of their parents, and once they had ‘settled’ the doctors and nurses were unaware of their distress. John Bowlby's interest in maternal deprivation led him to appoint James Robertson as his research assistant, to observe responses of young children to loss of maternal care on admission to hospital. They formulated the theoretical framework of the three stages through which the children went; protest, despair, and detachment constituting a developmental interference. Robertson was so concerned when nobody would listen that in 1952 he made the film ‘A Two Year Old Goes to Hospital’, which upset children's doctors and nurses. It also probably contributed to the government setting up in 1957 a Committee chaired by Sir Harry Platt to consider the Welfare of Children in Hospital. ‘Going to Hospital with Mother’ was made by Robertson in 1958. With Dermod MacCarthy he showed the films to the Committee, who accepted the suggestions in Robertson's Memorandum which included unrestricted visiting and mothers being admitted with their young children. The Report, known as the Platt Report, was published in 1959. Robertson could then show his films publicy, campaign in the media and encourage the pressure group NAWCH (the National Association for the Welfare of Children in Hospital) who were successful in getting many of the Committee's recommendations implemented, to the benefit of all children in hospital.     

Comments on “Changing attitudes towards the care of children in hospital: a new assessment of the influence of the work of Bowlby and Robertson in the UK, 1940–1970” by Frank C.P. van der Horst and Rene van der Veer (Attachment & Human Development Vol 11, No 2, March 2009, 119–142)

The importance of James Robertson's work in changing the hospital conditions for young children is widely acknowledged but disputed by van der Horst and van der Veer (2009), although they presented evidence supporting his influence, they also presented considerable evidence showing that others were ignored, including writers in The Lancet and the British Medical Journal. van der Horst and van der Veer presented a muddled picture by failing to understand how the work of James Robertson influenced a wide range of people, opened up the debate and, through the Platt Report, gave parents the authority and confidence to challenge hospitals and stay with their young children. James Robertson's 1952 film “a Two-year-old Goes to Hospital” provided the ‘visual communication to pierce defences as the spoken word cannot do’. This film continues to be widely used today in teaching about young children.     

Dengue fever virus and Japanese encephalitis virus synthetic peptides,with motifs to fit HLA class I haplotypes prevalent in human populations in endemic regions,can be used for application to skin Langerhans cells to prime antiviral CD8+ cytotoxic T cells (CTLs)—A novel approach to the protection of humans

Flaviviruses were reported to induce CD8⁺ cytotoxic T cells in infected individuals, indicating that nonapeptides, proteolytic cleavage products of the viral precursor protein, enter the endoplasmic reticulum in infected cells and interact with HLA class I molecules. The assembled HLA class I molecules are transported to the plasma membrane and prime CD8⁺ T cells. Current knowledge of the interaction of viral peptides with HLA molecules is reviewed. Based on this review, an idea is presented to use synthetic flavivirus peptides with an amino acid motif to fit with the HLA class I peptide binding group of HLA haplotypes prevalent in a given population in an endemic area. These synthetic viral peptides may be introduced into the human skin using a lotion containing the peptides (Peplotion) together with substances capable of enhancing the penetration of these peptides into the skin to reach Langerhans cells. The peptide-treated Langerhans cells, professional antigen-presenting cells, may bind the synthetic viral peptides by their HLA class I peptide-binding grooves. Antigens carrying Langerhans cells are able to migrate and induce the cellular immune response in the lymph nodes. This approach to the priming of antiviral CD8⁺ cytotoxic T cells may provide cellular immune protection from flavivirus infection without inducing the humoral immune response, which can lead to the shock syndrome in Dengue fever patients. To be able to develop anti-Dengue virus synthetic peptides for populations with different HLA class I haplotypes, it is necessary to develop computational studies to design HLA class I Dengue virus synthetic peptides with motifs to fit the HLA haplotypes of the population living in an endemic region for Dengue fever. Experiments to study Dengue virus and Japanese encephalitis peptides vaccines and their effectiveness in protection against Dengue fever and Japanese encephalitis are needed. The development of human antiviral vaccines for application of viral peptides in a lotion to human skin (Peplotion) may be useful and affordable for populations of developing countries.