IL-18 inhibits growth of murine orthotopic prostate carcinomas via both adaptive and innate immune mechanisms

Interleukin(IL)-18 is a pleiotrophic cytokine with functions in immune modulation, angiogenesis and bone metabolism. In this study, the potential of IL-18 as an immunotherapy for prostate cancer (PCa) was examined using the murine model of prostate carcinoma, RM1 and a bone metastatic variant RM1(BM)/B4H7-luc. RM1 and RM1(BM)/B4H7-luc cells were stably transfected to express bioactive IL-18. These cells were implanted into syngeneic immunocompetent mice, with or without an IL-18-neutralising antibody (αIL-18, SK113AE4). IL-18 significantly inhibited the growth of both subcutaneous and orthotopic RM1 tumors and the IL-18 neutralizing antibody abrogated the tumor growth-inhibition. In vivo neutralization of interferon-gamma (IFN-γ) completely eliminated the anti-tumor effects of IL-18 confirming an essential role of IFN-γ as a down-stream mediator of the anti-tumor activity of IL-18. Tumors from mice in which IL-18 and/or IFN-γ was neutralized contained significantly fewer CD4+ and CD8+ T cells than those with functional IL-18. The essential role of adaptive immunity was demonstrated as tumors grew more rapidly in RAG1−/− mice or in mice depleted of CD4+ and/or CD8+ cells than in normal mice. The tumors in RAG1−/− mice were also significantly smaller when IL-18 was present, indicating that innate immune mechanisms are involved. IL-18 also induced an increase in tumor infiltration of macrophages and neutrophils but not NK cells. In other experiments, direct injection of recombinant IL-18 into established tumors also inhibited tumor growth, which was associated with an increase in intratumoral macrophages, but not T cells. These results suggest that local IL-18 in the tumor environment can significantly potentiate anti-tumor immunity in the prostate and clearly demonstrate that this effect is mediated by innate and adaptive immune mechanisms.

Defining a 3-dimensional (3D) in vitro model to study immune cell and renal cell interactions

This study aimed to develop a 3-Dimensional (D) hydrogel system for the co-culture of autologous human renal and immune cells. Previous studies have shown that human renal epithelial cells are able to modulate autologous immune cell responses. However, these studies were undertaken in a standard 2D culture system. The 3D model was developed to re-capitulate these observations within a more physiological relevant in vivo like environment.

The mechanisms of human renal epithelial cell modulation of autologous dendritic cell phenotype and function

Proximal tubule epithelial cells (PTEC) of the kidney line the proximal tubule downstream of the glomerulus and play a major role in the re-absorption of small molecular weight proteins that may pass through the glomerular filtration process. In the perturbed disease state PTEC also contribute to the inflammatory disease process via both positive and negative mechanisms via the production of inflammatory cytokines which chemo-attract leukocytes and the subsequent down-modulation of these cells to prevent uncontrolled inflammatory responses. It is well established that dendritic cells are responsible for the initiation and direction of adaptive immune responses. Both resident and infiltrating dendritic cells are localised within the tubulointerstitium of the renal cortex, in close apposition to PTEC, in inflammatory disease states. We previously demonstrated that inflammatory PTEC are able to modulate autologous human dendritic cell phenotype and functional responses. Here we extend these findings to characterise the mechanisms of this PTEC immune-modulation using primary human PTEC and autologous monocyte-derived dendritic cells (MoDC) as the model system. We demonstrate that PTEC express three inhibitory molecules: (i) cell surface PD-L1 that induces MoDC expression of PD-L1; (ii) intracellular IDO that maintains the expression of MoDC CD14, drives the expression of CD80, PD-L1 and IL-10 by MoDC and inhibits T cell stimulatory capacity; and (iii) soluble HLA-G (sHLA-G) that inhibits HLA-DR and induces IL-10 expression by MoDC. Collectively the results demonstrate that primary human PTEC are able to modulate autologous DC phenotype and function via multiple complex pathways. Further dissection of these pathways is essential to target therapeutic strategies in the treatment of inflammatory kidney disorders.

Human proximal tubule epithelial cells modulate autologous dendritic cell function

Background We have previously demonstrated that human kidney proximal tubule epithelial cells (PTEC) are able to modulate autologous T and B lymphocyte responses. It is well established that dendritic cells (DC) are responsible for the initiation and direction of adaptive immune responses and that these cells occur in the renal interstitium in close apposition to PTEC under inflammatory disease settings. However, there is no information regarding the interaction of PTEC with DC in an autologous human context. Methods Human monocytes were differentiated into monocyte-derived DC (MoDC) in the absence or presence of primary autologous activated PTEC and matured with polyinosinic:polycytidylic acid [poly(I:C)], while purified, pre-formed myeloid blood DC (CD1c+ BDC) were cultured with autologous activated PTEC in the absence or presence of poly(I:C) stimulation. DC responses were monitored by surface antigen expression, cytokine secretion, antigen uptake capacity and allogeneic T-cell-stimulatory ability. Results The presence of autologous activated PTEC inhibited the differentiation of monocytes to MoDC. Furthermore, MoDC differentiated in the presence of PTEC displayed an immature surface phenotype, efficient phagocytic capacity and, upon poly(I:C) stimulation, secreted low levels of pro-inflammatory cytokine interleukin (IL)-12p70, high levels of anti-inflammatory cytokine IL-10 and induced weak Th1 responses. Similarly, pre-formed CD1c+ BDC matured in the presence of PTEC exhibited an immature tolerogenic surface phenotype, strong endocytic and phagocytic ability and stimulated significantly attenuated T-cell proliferative responses. Conclusions Our data suggest that activated PTEC regulate human autologous immunity via complex interactions with DC. The ability of PTEC to modulate autologous DC function has important implications for the dampening of pro-inflammatory immune responses within the tubulointerstitium in renal injuries. Further dissection of the mechanisms of PTEC modulation of autologous immune responses may offer targets for therapeutic intervention in renal medicine.

Effect of age, gender and mannose-binding lectin (MBL) status on the inflammatory profile in peripheral blood plasma of Australian blood donors

Transfusion of blood components has been associated with poor patient outcomes and, an overall increase in morbidity and mortality. Differences in the blood components arising from donor health, age and immune status may impact on outcomes of transfusion and transfusion-related immune modulation in recipients. The aim of this study was to investigate differences in inflammatory profile in donors and association with parameters including age, gender and deficiency status of pattern recognition molecule mannose-binding lectin (MBL). MBL level was determined by ELISA. Serum levels of interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-10, IL-12, tumour necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1α, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-α, and IFN-γ were examined by cytometric bead array (CBA). C-reactive protein (CRP) and rheumatoid factor (RF) were examined by immunoturbidimetry. This study demonstrated age was a parameter associated with the immune profile of blood donors, with significant increases in MCP-1 (p < 0.05) and RF (p < 0.05) and decreases in IL-1α evident in the older donors (61–76 years). Significant gender-associated differences in MCP-1, IL-12 and CRP plasma levels in the blood donor cohort were also reported. There was no significant difference in the level of any inflammatory markers studied according to MBL status. This study demonstrated that age and gender are associated with inflammatory profile in donors. These differences may be a factor impacting on outcomes of transfusion.

Modulation of the Chlamydia trachomatis In vitro transcriptome response by the sex hormones estradiol and progesterone

BACKGROUND:Chlamydia trachomatis is a major cause of sexually transmitted disease in humans. Previous studies in both humans and animal models of chlamydial genital tract infection have suggested that the hormonal status of the genital tract epithelium at the time of exposure can influence the outcome of the chlamydial infection. We performed a whole genome transcriptional profiling study of C. trachomatis infection in ECC-1 cells under progesterone or estradiol treatment.RESULTS:Both hormone treatments caused a significant shift in the sub-set of genes expressed (25% of the transcriptome altered by more than 2-fold). Overall, estradiol treatment resulted in the down-regulation of 151 genes, including those associated with lipid and nucleotide metabolism. Of particular interest was the up-regulation in estradiol-supplemented cultures of six genes (omcB, trpB, cydA, cydB, pyk and yggV), which suggest a stress response similar to that reported previously in other models of chlamydial persistence. We also observed morphological changes consistent with a persistence response. By comparison, progesterone supplementation resulted in a general up-regulation of an energy utilising response.CONCLUSION:Our data shows for the first time, that the treatment of chlamydial host cells with key reproductive hormones such as progesterone and estradiol, results in significantly altered chlamydial gene expression profiles. It is likely that these chlamydial expression patterns are survival responses, evolved by the pathogen to enable it to overcome the host's innate immune response. The induction of chlamydial persistence is probably a key component of this survival response.

Induction of a Th1 immune response and suppression of IgE via immunotherapy with a recombinant hybrid molecule encapsulated in liposome-protamine-DNA nanoparticles in a model of experimental allergy

Liposome-protamine-DNA nanoparticles (LPD) are safe, effective, and non-toxic adjuvants that induce Th1-like immune responses. We hypothesized that encapsulation of allergens into liposomes could be an appropriate option for immunotherapy. The present study evaluated the immunotherapeutic potential of a recombinant hybrid molecule (rHM) encapsulated in LPD nanoparticles in a murine model of Chenopodium album allergy. BALB/c mice were sensitized with the allergen in alum, and the immunotherapy procedure was performed by subcutaneous injections of LPD-rHM, rHM, or empty LPD at weekly intervals. Sensitized mice developed a Th2-biased immune response characterized by strong specific IgG1 and IgE production, IL-4, and the transcription factor GATA3 in spleen cell cultures. Treatment with the LPD-rHM resulted in a reduction in IgE and a marked increase in IgG2a. The LPD-rHM induced allergen-specific responses with relatively high interferon-gamma production, as well as expression of the transcription factor T-bet in stimulated splenocytes. In addition, lymphoproliferative responses were higher in the LPD-rHM-treated mice than in the other groups. Removal of the nanoparticles from the rHM resulted in a decrease in the allergen's immunogenicity. These results indicate that the rHM complexed with LPD nanoparticles has a marked suppressive effect on the allergic response and caused a shift toward a Th1 pathway.

Modulation of the SHBG signalling axis

Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein that is the major sex steroid carrier-protein in the bloodstream and functions also as a key regulator of steroid bioavailability within target tissues, such as the prostate. Additionally, SHBG binds to prostatic cell membranes via the putative and unidentified SHBG receptor (RSHBG), activating a signal transduction pathway implicated in stimulating both proliferation and expression of prostate specific antigen (PSA) in prostate cell lines in vitro. A yeast-two hybrid assay suggested an interaction between SHBG and kallikrein-related protease (KLK) 4, which is a serine protease implicated in the progression of prostate cancer. The potential interaction between these two proteins was investigated in this PhD thesis to determine whether SHBG is a proteolytic substrate of KLK4 and other members of the KLK family including KLK3/PSA, KLK7 and KLK14. Furthermore, the effects from SHBG proteolytic degradation on SHBG-regulated steroid bioavailability and the activation of the putative RSHBG signal transduction pathway were examined in the LNCaP prostate cancer cell line. SHBG was found to be a proteolytic substrate of the trypsin-like KLK4 and KLK14 in vitro, yielding several proteolysis fragments. Both chymotrypsin-like PSA and KLK7 displayed insignificant proteolytic activity against SHBG. The kinetic parameters of SHBG proteolysis by KLK4 and KLK14 demonstrate a strong enzyme-substrate binding capacity, possessing a Km of 1.2 ± 0.7 µM and 2.1 ± 0.6 µM respectively. The catalytic efficiencies (kcat/Km) of KLK4 and KLK14 proteolysis of SHBG were 1.6 x 104 M-1s-1 and 3.8 x 104 M-1s-1 respectively, which were comparable to parameters previously reported for peptide substrates. N-terminal sequencing of the fragments revealed cleavage near the junction of the N- and C-terminal laminin globulin-like (G-like) domains of SHBG, resulting in the division of the two globulins and ultimately the full degradation of these fragments by KLK4 and KLK14 over time. Proteolytic fragments that may retain steroid binding were rapidly degraded by both proteases, while fragments containing residues beyond the steroid binding pocket were less degraded over the same period of time. Degradation of SHBG was inhibited by the divalent metal cations calcium and zinc for KLK4, and calcium, zinc and magnesium for KLK14. The human secreted serine protease inhibitors (serpins), α1-antitrypsin and α2-antiplasmin, inhibited KLK4 and KLK14 proteolysis of SHBG; α1-antichymotrypsin inhibited KLK4 but not KLK14 activity. The inhibition by these serpins was comparable and in some cases more effective than general trypsin protease inhibitors such as aprotinin and phenylmethanesulfonyl fluoride (PMSF). The binding of 5α-dihydrotestosterone (DHT) to SHBG modulated interactions with KLK4 and KLK14. Steroid-free SHBG was more readily digested by both enzymes than DHT-bound SHBG. Moreover, a binding interaction exists between SHBG and pro-KLK4 and pro-KLK14, with DHT strengthening the binding to pro-KLK4 only. The inhibition of androgen uptake by cultured prostate cancer cells, mediated by SHBG steroid-binding, was examined to assess whether SHBG proteolysis by KLK4 and KLK14 modulated this process. Proteolytic digestion eliminated the ability of SHBG to inhibit the uptake of DHT from conditioned media into LNCaP cells. Therefore, the proteolysis of SHBG by KLK4 and KLK14 increased steroid bioavailability in vitro, leading to an increased uptake of androgens by prostate cancer cells. Interestingly, different transcriptional responses of PSA and KLK2, which are androgen-regulated genes, to DHT-bounsd SHBG treatment were observed between low and high passage number LNCaP cells (lpLNCaP and hpLNCaP respectively). HpLNCaP cells treated with DHT-bound SHBG demonstrated a significant synergistic upregulation of PSA and KLK2 above DHT or SHBG treatment alone, which is similar to previously reported downstream responses from RSHBG-mediated signaling activation. As this result was not seen in lpLNCaP cells, only hpLNCaP cells were further investigated to examine the modulation of potential RSHBG activity by KLK4 and KLK14 proteolysis of SHBG. Contrary to reported results, no increase in intracellular cAMP was observed in hpLNCaP cells when treated with SHBG in the presence and absence of either DHT or estradiol. As a result, the modulation of RSHBG-mediated signaling activation could not be determined. Finally, the identification of the RSHBG from both breast (MCF-7) and prostate cancer (LNCaP) cell lines was attempted. Fluorescently labeled peptides corresponding to the putative receptor binding domain (RBD) of SHBG were shown to be internalized by MCF-7 cells. Crosslinking of the RBD peptide to the cell surfaces of both MCF-7 and LNCaP cells, demonstrated the interaction of the peptide with several targets. These targets were then captured using RBD peptides synthesized onto a hydrophilic scaffold and analysed by mass spectrometry. The samples captured by the RBD peptide returned statistically significantly matches for cytokeratin 8, 18 and 19 as well as microtubule-actin crosslinking factor 1, which may indicate a novel interaction between SHBG and these proteins, but ultimately failed to detect a membrane receptor potentially responsible for the putative RSHBG-mediated signaling. This PhD project has reported the proteolytic processing of SHBG by two members of the kallikrein family, KLK4 and KLK14. The effect of SHBG proteolysis by KLK4 and KLK14 on RSHBG-mediated signaling activation was unable to be determined as the reported signal transduction pathway was not activated after treatment with SHBG, in combination with either DHT or estradiol. However, the digestion of SHBG by these two proteases positively regulated androgen bioavailability to prostate cancer cells in vitro. The increased uptake of androgens is deleterious in prostate cancer due to the promotion of proliferation, metastasis, invasion and the inhibition of apoptosis. The increased bioavailability of androgens, from SHBG proteolysis by KLK4 and KLK14, may therefore promote both carcinogenesis and progression of prostate cancer. Finally, this information may contribute to the development of therapeutic treatment strategies for prostate cancer by inhibiting the proteolysis of SHBG, by KLK4 and KLK14, to prevent the increased uptake of androgens by hormone-dependent cancerous tissues.

Characteristic analysis for multisampled digital implementation of fixed-switching-frequency closed-loop modulation of voltage-source inverter

In this paper, a fixed-switching-frequency closed-loop modulation of a voltage-source inverter (VSI), upon the digital implementation of the modulation process, is analyzed and characterized. The sampling frequency of the digital processor is considered as an integer multiple of the modulation switching frequency. An expression for the determination of the modulation design parameter is developed for smooth modulation at a fixed switching frequency. The variation of the sampling frequency, switching frequency, and modulation index has been analyzed for the determination of the switching condition under closed loop. It is shown that the switching condition determined based on the continuous-time analysis of the closed-loop modulation will ensure smooth modulation upon the digital implementation of the modulation process. However, the stability properties need to be tested prior to digital implementation as they get deteriorated at smaller sampling frequencies. The closed-loop modulation index needs to be considered maximum while determining the design parameters for smooth modulation. In particular, a detailed analysis has been carried out by varying the control gain in the sliding-mode control of a two-level VSI. The proposed analysis of the closed-loop modulation of the VSI has been verified for the operation of a distribution static compensator. The theoretical results are validated experimentally on both single- and three-phase systems.

Contemporary examples where doctors are not immune from non-fatal offences against a person

While it is uncontested that the medical profession makes a valuable contribution to society, doctors should not always be beyond the reach of the criminal law and they should not automatically be treated as God. Doctors should act reasonably and be conscious of their position of trust. In this sense, the notion of “doctors” is construed broadly to include a range of health care professionals such as podiatrists, radiographers, surgeons and general practitioners. This paper will explore contemporary Australian examples where doctors have acted inappropriately and been convicted of non-fatal offences against the person. The physical invasiveness involved in these scenarios varies significantly. In one example, a doctor penetrates a patient’s private body part with a probe for their own sexual gratification, and in another, a doctor covertly visually records a naked patient. The examples will be connected to the theories underpinning criminalisation, particularly social welfare and individual autonomy, with a view to framing guidelines on when doctors should not be immune from non-fatal offences against a person, and thus where the criminal law should respond.

The neuro-muscular and musculo-skeletal characterization of children with joint hypermobility

In children, joint hypermobility (typified by structural instability of joints) manifests clinically as neuro-muscular and musculo-skeletal conditions and conditions associated with development and organization of control of posture and gait (Finkelstein, 1916; Jahss, 1919; Sobel, 1926; Larsson, Mudholkar, Baum and Srivastava, 1995; Murray and Woo, 2001; Hakim and Grahame, 2003; Adib, Davies, Grahame, Woo and Murray, 2005:). The process of control of the relative proportions of joint mobility and stability, whilst maintaining equilibrium in standing posture and gait, is dependent upon the complex interrelationship between skeletal, muscular and neurological function (Massion, 1998; Gurfinkel, Ivanenko, Levik and Babakova, 1995; Shumway-Cook and Woollacott, 1995). The efficiency of this relies upon the integrity of neuro-muscular and musculo-skeletal components (ligaments, muscles, nerves), and the Central Nervous System’s capacity to interpret, process and integrate sensory information from visual, vestibular and proprioceptive sources (Crotts, Thompson, Nahom, Ryan and Newton, 1996; Riemann, Guskiewicz and Shields, 1999; Schmitz and Arnold, 1998) and development and incorporation of this into a representational scheme (postural reference frame) of body orientation with respect to internal and external environments (Gurfinkel et al., 1995; Roll and Roll, 1988). Sensory information from the base of support (feet) makes significant contribution to the development of reference frameworks (Kavounoudias, Roll and Roll, 1998). Problems with the structure and/ or function of any one, or combination of these components or systems, may result in partial loss of equilibrium and, therefore ineffectiveness or significant reduction in the capacity to interact with the environment, which may result in disability and/ or injury (Crotts et al., 1996; Rozzi, Lephart, Sterner and Kuligowski, 1999b). Whilst literature focusing upon clinical associations between joint hypermobility and conditions requiring therapeutic intervention has been abundant (Crego and Ford, 1952; Powell and Cantab, 1983; Dockery, in Jay, 1999; Grahame, 1971; Childs, 1986; Barton, Bird, Lindsay, Newton and Wright, 1995a; Rozzi, et al., 1999b; Kerr, Macmillan, Uttley and Luqmani, 2000; Grahame, 2001), there has been a deficit in controlled studies in which the neuro-muscular and musculo-skeletal characteristics of children with joint hypermobility have been quantified and considered within the context of organization of postural control in standing balance and gait. This was the aim of this project, undertaken as three studies. The major study (Study One) compared the fundamental neuro-muscular and musculo-skeletal characteristics of 15 children with joint hypermobility, and 15 age (8 and 9 years), gender, height and weight matched non-hypermobile controls. Significant differences were identified between previously undiagnosed hypermobile (n=15) and non-hypermobile children (n=15) in passive joint ranges of motion of the lower limbs and lumbar spine, muscle tone of the lower leg and foot, barefoot CoP displacement and in parameters of barefoot gait. Clinically relevant differences were also noted in barefoot single leg balance time. There were no differences between groups in isometric muscle strength in ankle dorsiflexion, knee flexion or extension. The second comparative study investigated foot morphology in non-weight bearing and weight bearing load conditions of the same children with and without joint hypermobility using three dimensional images (plaster casts) of their feet. The preliminary phase of this study evaluated the casting technique against direct measures of foot length, forefoot width, RCSP and forefoot to rearfoot angle. Results indicated accurate representation of elementary foot morphology within the plaster images. The comparative study examined the between and within group differences in measures of foot length and width, and in measures above the support surface (heel inclination angle, forefoot to rearfoot angle, normalized arch height, height of the widest point of the heel) in the two load conditions. Results of measures from plaster images identified that hypermobile children have different barefoot weight bearing foot morphology above the support surface than non-hypermobile children, despite no differences in measures of foot length or width. Based upon the differences in components of control of posture and gait in the hypermobile group, identified in Study One and Study Two, the final study (Study Three), using the same subjects, tested the immediate effect of specifically designed custom-made foot orthoses upon balance and gait of hypermobile children. The design of the orthoses was evaluated against the direct measures and the measures from plaster images of the feet. This ascertained the differences in morphology of the modified casts used to mould the orthoses and the original image of the foot. The orthoses were fitted into standardized running shoes. The effect of the shoe alone was tested upon the non-hypermobile children as the non-therapeutic equivalent condition. Immediate improvement in balance was noted in single leg stance and CoP displacement in the hypermobile group together with significant immediate improvement in the percentage of gait phases and in the percentage of the gait cycle at which maximum plantar flexion of the ankle occurred in gait. The neuro-muscular and musculo-skeletal characteristics of children with joint hypermobility are different from those of non-hypermobile children. The Beighton, Solomon and Soskolne (1973) screening criteria successfully classified joint hypermobility in children. As a result of this study joint hypermobility has been identified as a variable which must be controlled in studies of foot morphology and function in children. The outcomes of this study provide a basis upon which to further explore the association between joint hypermobility and neuro-muscular and musculo-skeletal conditions, and, have relevance for the physical education of children with joint hypermobility, for footwear and orthotic design processes, and, in particular, for clinical identification and treatment of children with joint hypermobility.