Tobacco Constituents, Flavorants, and Paper Permeability of Factory-Made and Roll-Your-Own Cigarettes on the Australian Market.

INTRODUCTION: Roll-your-own (RYO) tobacco is a popular choice in Australia, with some people who smoke finding these products more attractive than factory-made cigarettes (FMC). Differences in visual and tactile properties and in the feel and taste of the smoke may contribute to this attractiveness. These differences may be driven by variation in tobacco constituents and wrapping paper permeability. However, to date, there has been no comparison of RYO and FMC products on the Australian market. AIMS AND METHODS: Chemical constituents, pH, flavorants, and paper permeability were compared in unburned RYO tobacco and tobacco from FMC. RYO and FMC products from matched brands were compared, as were products from the most popular FMC and RYO brands on the Australian market in 2018. RESULTS: RYO tobacco had higher moisture and humectant content (glycerol and propylene glycol) than FMC tobacco. RYO tobacco also had higher amounts of total and reducing sugars and lower nicotine when comparing the most popular brands. RYO papers were less permeable than FMC papers. Both RYO and FMC tobacco contained many chemicals identified as flavorants, including fourteen with known potential health risks. For most measured constituents and flavorants, RYO tobaccos had more in common with other RYO than FMC, with the commonalities remaining even when matched brands were compared. CONCLUSIONS: Higher levels of moisture, humectants, and sugars in Australian RYO tobacco compared to FMC may be increasing attractiveness of RYO by reducing the harsh taste of the smoke and increasing the moist feel of the tobacco. IMPLICATIONS: While price is the main factor driving the use of RYO tobacco, some people who smoke find these products more attractive. This study has shown that Australian RYO tobacco contains higher amounts of glycerol, propylene glycol, and sugars than FMC. These chemicals may be improving the taste of the tobacco, as well as creating a moist feel that is falsely perceived as indicating that the tobacco is "fresh" and "less chemically." Ironically, it may be that higher amounts of some added chemicals in RYO contribute to false perceptions of a more natural and less harmful product.

Simian immunodeficiency virus infection and immune responses in the pig-tailed macaque testis.

The testis is a site of immune privilege in rodents, and there is evidence that T cell responses are also suppressed in the primate testis. Local immunosuppression is a potential mechanism for HIV persistence in tissue reservoirs that few studies have examined. The response of the pig-tailed macaque testis to SIVmac239 infection was characterized to test this possibility. Testes were surgically removed during early-chronic (10 wk) and late-chronic (24-30 wk) SIV infection in 4 animals and compared with those from 7 uninfected animals. SIV infection caused only minor disruption to the seminiferous epithelium without marked evidence of inflammation or consistent changes in total intratesticular leukocyte numbers. Infection also led to an increase in the relative proportion of testicular effector memory CD8(+) T cell numbers and a corresponding reduction in central memory CD4(+) T cells. A decrease in the relative proportion of resident-type CD163(+) macrophages and DCs was also observed. SIV-specific CD8(+) T cells were detectable in the testis, 10-11 wk after infection by staining with SIV Gag-specific or Tat-specific MHC-I tetramers. However, testicular CD8(+) T cells from the infected animals had suppressed cytokine responses to mitogen activation. These results support the possibility that local immunosuppression in the testis may be restricting the ability of T cells to respond to SIV or HIV infection. Local immunosuppression in the testis may be an underexplored mechanism allowing HIV persistence.

The maturation of antibody technology for the HIV epidemic.

Antibodies are one of our most useful biological tools. Indeed, improvements in antibody-based technologies have ushered in a new era of antibody-based therapeutics, research and diagnostic tools. Although improved technologies have led to the development of therapeutic antibodies for treatment of malignancies and inflammatory conditions, the use of advanced antibody technology in the therapy of viral infections is in its infancy. Non-human primate studies have demonstrated that antibodies against the HIV envelope can both prevent viral infection and control viremia. Despite the obvious potential of antibody therapies against HIV, there remain limitations in production and purification capacity that require further research. Recent advances in recombinant antibody technology have led to the development of a range of novel antibody fragments, such as single-domain nanobodies and bispecific antibodies, that are capable of targeting cancer cells to cytotoxic T cells. Novel antibody production techniques have also been designed, allowing antibodies to be obtained from non-mammalian cells, bovine colostrum and the periplasm and cytoplasm of bacteria. These advances may allow large-scale production of HIV antibodies that are capable of protecting against HIV infection or serving as therapeutics that reduce the need for life-long antiretroviral treatment. This review summarises recent advances in antibody-based technologies and discusses the possibilities and challenges of using these advances to design prophylactics and therapeutics against HIV.

Characterisation of macaque testicular leucocyte populations and T-lymphocyte immunity.

The rodent testis is well established as a site of immune privilege where both innate and acquired immune responses are suppressed. Immune cells and responses within human or non-human primate testes, by contrast, are poorly characterised. This study used multi-colour flow cytometry to characterise the leukocytes in testicular cells isolated from 12 young adult pigtail macaques (Macaca nemestrina) by collagenase dispersal, and to measure the cytokine responses of macaque testicular T-lymphocytes to mitogens. B-lymphocytes and granulocytes were present in very low numbers (0.24% and 3.3% of leukocytes respectively), indicating minimal blood contamination. A median of 30.8% of the recovered testicular leukocytes were CD3+ lymphocytes, with CD4 and CD8 T-lymphocyte proportions similar to those in the blood. The proportion of naïve T-lymphocytes in the testis was low, with significantly higher frequencies of central memory cells, compared with the blood. A median of 42.7% of the testicular leukocytes were CD163+ macrophages, while 4.5% were CD14+CD163- monocyte-like macrophages. Small populations of myeloid and plasmacytoid dendritic cells, NK cells and NKT cells were also detected. Following mitogen stimulation, 19.7% of blood T-lymphocytes produced IFNγ and/or TNF, whereas significantly fewer (4.4%) of the testicular T-lymphocytes responded to stimulation. Our results characterise the immune cells within the adult macaque testis and identify a suppression of T-lymphocyte responses. This study provides a baseline to examine the immunology of the primate testis and suggests that testicular immune privilege could also be present in primates.

Phenotypic and functional heterogeneity of the testicular macrophage population: a new regulatory model.

Testicular macrophages (TMs) are important contributors to the response of the testis to infection, as well as the regulation of spermatogenesis, steroidogenesis and other homeostatic functions of the testis. The TMs are the largest population of immune cells in a region of tight immunoregulation, where both innate and acquired immune responses are effectively suppressed, and these cells are predicted to be responsible for regulating this immunosuppression. In the rat, TMs have been broadly classified into two main populations, designated "newly arrived" and "resident", the latter being characterised by expression of the scavenger receptor, CD163. Systemic inflammation in response to lipopolysaccharide leads to an influx of CD163(-) monocyte-like ("infiltrating") macrophages into the rodent testis, which have a pro-inflammatory phenotype and express interleukin-1ß, tumour necrosis factor-α, inducible nitric oxide synthase and other inflammatory factors. The resident CD163(+) TMs, on the other hand, constitutively produce IL-10 and are poor stimulators of T-cell proliferation in vitro, indicating that they contribute to testicular immunosuppression. However, our recent studies have demonstrated that the "newly-arrived" CD163(-) TMs present in the rat testis under normal homeostatic conditions show very little response to LPS stimulation in vitro. We here propose a modified model of TM heterogeneity whereby the CD163(-) TMs of the normal rat testis are derived from a monocyte subset that continuously repopulates the testis, and is distinct from the monocyte-like "infiltrating" subset from which pro-inflammatory CD163(-) TMs may be derived during systemic inflammation.

Regulation of activin A release from murine bone marrow-derived neutrophil precursors by tumour necrosis factor-α and insulin.

Activin A, a transforming growth factor-ß family cytokine, plays a crucial role in regulating the onset and severity of many inflammatory conditions, such as acute lipopolysaccharide (LPS)-induced inflammation. Activin A is also implicated in type 2 diabetes (T2D), a disease characterised by insulin resistance, hyperglycaemia and chronic elevation of pro-inflammatory cytokines, including tumour necrosis factor (TNF-α). In the human, neutrophils contain activin A that can be released in response to TNF-α. Studies of inflammatory disease in vivo, however, generally use the mouse, so it is essential to know if murine neutrophils have similar properties. Regulation of activin A was investigated in bone marrow-derived neutrophil precursors (BMNPs) from 8 to 10 weeks old C57BL6/J male mice. The BMNPs contained 7-fold higher concentrations of activin A than bone marrow mononuclear cells. Release of activin A from isolated BMNPs was stimulated by TNF-α, but this was not due to increased activin A production. In contrast to TNF-α, LPS had no effect on isolated BMNPs, but stimulated activin A release and production in total bone marrow cell cultures. Moreover, activin A release in response to LPS, was not prevented in TNF-α null mice. Increased glucose and insulin had no effect on base-line activin A secretion by BMNPs in culture, but pre-treatment with insulin blocked the TNF-α induced release of activin A. These results indicate that murine neutrophils are a source of stored activin A, the release of which can be directly stimulated by TNF-α, although TNF-α is not the only stimulator of activin A release during inflammation. Furthermore, regulation of neutrophil activin A release by insulin may also play a role in the inflammation associated with T2D.

Rat resident testicular macrophages have an alternatively activated phenotype and constitutively produce interleukin-10 in vitro.

The ability of the rodent testis to tolerate graft alloantigens and spermatogenic cell autoantigens is well known. The mechanisms underlying this "immune privilege" are poorly understood, but the numerous resident TMs have been implicated. Although it has been assumed that TMs display a phenotype consistent with immune privilege, this has not been formally established. Consequently, TMs were isolated from adult rats and cultured under basal conditions and following stimulation with LPS and IFN-γ (classical activation) or IL-4 (alternative activation). BMMs matured in vitro were used as control. Expression of the classical (proinflammatory) activation markers TNF-α, IL-1ß, iNOS, IL-6, RANTES, IL-12p40, and SOCS3 and alternative (immunoregulatory) activation markers IL-10, TGF-ß1, CXCL2, and SOCS1 was measured by QPCR or ELISA. In culture, TMs were characterized by poor expression of classical activation genes and TGF-ß1 but constitutively high IL-10 production and reduced costimulatory activity in a polyclonal T cell activation assay. This pattern of gene expression was associated with TMs expressing the scavenger receptor CD163, which is characteristic of tissue resident macrophages and alternative activation. By contrast, CD163-negative TMs displayed reduced inflammatory gene expression but did not constitutively produce IL-10. These data indicate that under the influence of the testicular environment, macrophages adopt an alternatively activated phenotype, involving reduced capacity for proinflammatory gene expression, constitutive IL-10 production, and impaired ability to support T cell activation, consistent with a role in maintaining testicular immune privilege.

The regulation and functions of activin and follistatin in inflammation and immunity.

The activins are members of the transforming growth factor ß superfamily with broad and complex effects on cell growth and differentiation. Activin A has long been known to be a critical regulator of inflammation and immunity, and similar roles are now emerging for activin B, with which it shares 65% sequence homology. These molecules and their binding protein, follistatin, are widely expressed, and their production is increased in many acute and chronic inflammatory conditions. Synthesis and release of the activins are stimulated by inflammatory cytokines, Toll-like receptor ligands, and oxidative stress. The activins interact with heterodimeric serine/threonine kinase receptor complexes to activate SMAD transcription factors and the MAP kinase signaling pathways, which mediate inflammation, stress, and immunity. Follistatin binds to the activins with high affinity, thereby obstructing the activin receptor binding site, and targets them to cell surface proteoglycans and lysosomal degradation. Studies on transgenic mice and those with gene knockouts, together with blocking studies using exogenous follistatin, have established that activin A plays critical roles in the onset of cachexia, acute and chronic inflammatory responses such as septicemia, colitis and asthma, and fibrosis. However, activin A also directs the development of monocyte/macrophages, myeloid dendritic cells, and T cell subsets to promote type 2 and regulatory immune responses. The ability of both endogenous and exogenous follistatin to block the proinflammatory and profibrotic actions of activin A has led to interest in this binding protein as a potential therapeutic for limiting the severity of disease and to improve subsequent damage associated with inflammation and fibrosis. However, the ability of activin A to sculpt the subsequent immune response as well means that the full range of effects that might arise from blocking activin bioactivity will need to be considered in any therapeutic applications.

Tumour necrosis factor-α stimulates human neutrophils to release preformed activin A.

Activin A, a member of the transforming growth factor-ß superfamily, is a critical early mediator of acute inflammation. Activin A release coincides with the release of tumour necrosis factor-α (TNF-α) in models of lipopolysaccharide (LPS)-induced inflammation. The source of circulating activin A during acute inflammation has not been identified and the potential contribution of leukocyte subsets was examined in the following study. Human leukocytes from healthy volunteers were fractionated using Ficoll gradients and cultured under serum-free conditions. Freshly isolated human neutrophils contained 20-fold more activin A than blood mononuclear cells as measured by enzyme-linked immunosorbent assay (ELISA), and both dimeric and monomeric forms of activin A were detected in these cells by western blotting. Activin A was predominantly immunolocalized in the neutrophil cytoplasm. Purified neutrophils secreted activin A in culture when stimulated by TNF-α, but were unable to respond to LPS directly. Although TNF-α stimulated activin A release from neutrophils within 1 h, activin subunit mRNA expression did not increase until 12 h of culture, and the amount of activin A released following TNF-α stimulation did not change between 1 and 12 h. Specific inhibition of the p38 MAP kinase signalling pathway blocked TNF-α-induced activin release, and the secretion of activin A was not due to TNF-α-induced neutrophil apoptosis. These data provide the first evidence that neutrophils are a significant source of mature, stored activin A. Stimulation of the release of neutrophil activin A by TNF-α may contribute to the early peak in circulating activin A levels during acute inflammation.

Regulation of Sertoli cell activin A and inhibin B by tumour necrosis factor α and interleukin 1α: interaction with follicle-stimulating hormone/adenosine 3',5'-cyclic phosphate signalling.

Regulation of crucial events during spermatogenesis involves dynamic changes in cytokine production and interactions across the cycle of the seminiferous epithelium. Regulation of activin A and inhibin B production by the inflammatory cytokines, tumour necrosis factor α (TNFα) and interleukin 1α (IL1α), alone and in conjunction with FSH or a cAMP analogue (dibutyryl cAMP), was examined in cultures of Sertoli cells from 20-day old rats. Both TNFα and IL1α stimulated activin A secretion and expression of its subunit (ß(A)) mRNA, and suppressed inhibin B secretion and expression of its subunit (α and ß(B)) mRNAs. The actions of TNFα and IL1α were opposed by FSH and dibutyryl cAMP. Both cytokines inhibited FSH/dibutyryl cAMP-stimulated inhibin B secretion and mRNA expression as well as stem cell factor mRNA expression. Both cytokines also inhibited FSH-induced cAMP production, and reduced baseline FSH receptor mRNA expression. These data highlight the reciprocal relationship that exists between FSH/cAMP signalling and inflammatory cytokine signalling pathways in the control of Sertoli cell function, and production of activin A/inhibin B in particular. It is anticipated that these interactions play important roles in the fine control of events during the cycle of the seminiferous epithelium and in the inhibition of spermatogenesis during inflammation.

Differential responses of epithelial Sertoli cells of the rat testis to Toll-like receptor 2 and 4 ligands: implications for studies of testicular inflammation using bacterial lipopolysaccharides.

The relative contribution of epithelial Sertoli cells in response to bacterial infection of the testis remains poorly characterised, since studies on inflammatory properties of these cells have invariably used unpurified lipopolysaccharide (LPS) preparations contaminated with bacterial lipopeptides. Consequently, isolated rat Sertoli cells were stimulated with either unextracted or phenol re-extracted LPS, and analysed for Toll-like receptor (TLR) 4, TLR2 and inflammatory cytokine gene expression by quantitative reverse transcription polymerase chain reaction (RT-PCR). Expression of TLR4 and its co-receptor protein myeloid differentiation (MD) 2 in Sertoli cells and testicular macrophages were similar, but Sertoli cells displayed low basal or LPS-induced expression of the TLR4 accessory protein, CD14. In Sertoli cells, unextracted LPS produced cytokine responses which were considerably greater in magnitude and duration compared with their response to purified LPS. Sertoli cells also responded to the synthetic lipopeptide, Pam(3)Cys (a TLR2 ligand) with a similar pattern of prolonged gene expression. Sertoli cells were more than 10-fold less sensitive to purified LPS than macrophages, but expressed similar levels of interleukin (IL)-1α and IL-6, and much greater levels of the immunoregulatory cytokine activin A, when maximally stimulated. These data demonstrate that Sertoli cells display differential cytokine responses to bacterial stimuli, mediated by both TLR2 and TLR4, that are distinct from those of testicular macrophages.

Role of glutaredoxin1 and glutathione in regulating the activity of the copper-transporting P-type ATPases, ATP7A and ATP7B.

The copper-transporting P-type ATPases (Cu-ATPases), ATP7A and ATP7B, are essential for the regulation of intracellular copper homeostasis. In this report we describe new roles for glutathione (GSH) and glutaredoxin1 (GRX1) in Cu homeostasis through their regulation of Cu-ATPase activity. GRX1 is a thiol oxidoreductase that catalyzes the reversible reduction of GSH-mixed disulfides to their respective sulfhydryls (deglutathionylation). Here, we demonstrated that glutathionylation of the Cu-ATPases and their interaction with GRX1 were affected by alterations in Cu levels. The data support our hypothesis that the Cu-ATPases serve as substrates for Cu-dependent GRX1-mediated deglutathionylation. This in turn liberates the Cu-ATPase cysteinyl thiol groups for Cu binding and transport. GSH depletion experiments led to reversible inhibition of the Cu-ATPases that correlated with effects on intracellular Cu levels and GRX1 activity. Finally, knockdown of GRX1 expression resulted in an increase in intracellular Cu accumulation. Together, these data directly implicate GSH and GRX1 with important new roles in redox regulation of the Cu-ATPases, through modulation of Cu binding by the Cu-ATPase cysteine motifs.

Effects of chronic celecoxib on testicular function in normal and lipopolysaccharide-treated rats.

Celecoxib (Celebrex), an inhibitor of cyclooxygenase-2 (COX-2; prostaglandin-endoperoxide synthase 2; EC 1.14.99.1), is widely used in the treatment of chronic inflammation and pain. COX-2 is constitutively expressed in the testis, where it is responsible for prostaglandin production, so inhibition of this enzyme should have effects on testicular function. The effects of administering celecoxib (oral with feed, 0.15% w/w) for 5 weeks on normal testis function and the response to low dose (0.1 mg/kg body weight) or high dose (5.0 mg/kg) lipopolysaccharide (LPS) were examined in adult male rats. Celecoxib caused a 60% reduction in testicular interstitial fluid (IF) prostaglandin E(2) (PGE(2)) concentrations, accompanied by a compensatory increase in COX-2 mRNA expression. Celecoxib increased IF volume by 30%, but had no effect on testis weight, testis morphology or serum testosterone levels. In the celecoxib-fed rats, the dose-dependent inhibitory effects of LPS on testis weight, IF volume and serum testosterone levels were significantly diminished. However, celecoxib had no effect on COX-2 protein levels or LPS-induced expression of the inflammatory mediators interleukin-1beta, tumour necrosis factor-alpha or inducible nitric-oxide synthase. A similar lack of inhibition of LPS-induced cytokine expression by another COX-2 inhibitor, NS-398, was observed in vitro. These data indicate that celecoxib reduces intratesticular activity of COX-2 (as indicated by PGE(2) levels) and inhibits IF formation in the testis, but has no appreciable effect on steroidogenesis or spermatogenesis, at least in the short term. Celecoxib does not appear to alter the ability of the testis to mount an inflammatory response but opposes the deleterious effects of inflammation on IF formation and testosterone production. These results indicate significant roles for products of the COX-2 pathway in testicular vascular control and steroidogenesis, which may have implications for men with marginal fertility taking celecoxib for extended periods, but also highlight the potential of this drug to ameliorate testicular damage caused by systemic or local inflammation.