A variety of extraimmune system factors, including hormones, play a critical role in regulating immunity. Progesterone has been shown to affect immunity in rodents and humans, mainly at ...concentrations commensurate with pregnancy. These effects are primarily mediated via the progesterone receptor (PR), which acts as a transcription factor, although non-genomic effects of PR activation have been reported. In this study, we evaluated the effects of progesterone on rat dendritic cells (DCs) at ranges encompassing physiologic and pharmacologic concentrations to determine whether progesterone plays a role in modulating DC-mediated immune responses. DCs were derived by culturing rat bone marrow cells in granulocyte macrophage colony-stimulating factor and IL-4. Cells were analyzed for expression of PR using FACS analysis, real-time reverse transcriptase–PCR and fluorescent microscopy. Progesterone treatment of LPS-activated, mature bone marrow-derived dendritic cells (BMDCs) suppressed production of the pro-inflammatory response-promoting cytokines tumor necrosis factor-α and IL-1β in a dose-dependent manner but did not affect production of the pro-inflammatory response-inhibiting cytokine IL-10. Treatment of cells with progesterone also resulted in down-regulation of co-stimulatory molecule CD80 and MHC class II molecule RT1B expression. In addition, progesterone inhibited DC-stimulated proliferation of T cells. Suppression of pro-inflammatory response-promoting cytokine production by progesterone was prevented using the PR antagonist RU486. There was no dose-dependent effect of progesterone treatment on immature DC capacity to take up antigenic peptide. These data indicate that progesterone directly inhibits mature rat BMDC capacity to drive pro-inflammatory responses. This mechanism could contribute to or account for some of the differential expression of autoimmune/inflammatory disease in females.
During immune responses, antibodies are selected for their ability to bind to foreign
antigens with high affinity, in part by their ability to undergo homotypic bivalent
binding. However, this type ...of binding is not always possible. For example, the
small number of gp140 glycoprotein spikes displayed on the surface of the human
immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV,
somatic mutations that increase antibody affinity also increase breadth and
neutralizing potency. Surprisingly, the responding naive and memory B cells produce
polyreactive antibodies, which are capable of bivalent heteroligation between one
high-affinity anti-HIV-gp140 combining site and a second low-affinity site on
another molecular structure on HIV. Although cross-reactivity to self-antigens or
polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV,
Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134
monoclonal anti-HIV-gp140 antibodies cloned from six patients with
high titres of neutralizing antibodies are polyreactive. Despite the low affinity of
the polyreactive combining site, heteroligation demonstrably increases the apparent
affinity of polyreactive antibodies to HIV.
With better surgical outcomes, quicker recovery times, decreased postoperative pain, and reduced scarring at the surgical site, the application of minimally invasive surgery (MIS) has gained a lot of ...prominence in the last 30 years. This change in surgical practice has taken away the ability of a surgeon to palpate for the presence of a blood vessel as would occur in an open procedure. They instead must rely on a laparoscopic video camera feed that unfortunately cannot detect the presence of a blood vessel hidden beneath tissue. In certain scenarios, a surgeon can accidentally cut a blood vessel, which can lead to severe, even fatal, complications. Here, we show that by adding a near-infrared LED and a photodiode onto the opposing jaws of laparoscopic graspers, blood vessels buried under tissue can be detected. We show the results of Monte Carlo simulations to support our theory that the blood vessels ranging from 3 to 6 mm buried under up to 1 cm of tissue can be detected and quantified. This technology could be added to already existing laparoscopic tools that have limited surface areas on the jaws to assist surgeons during MIS procedures.
For centuries, surgeons have relied on their sense of touch to identify vital structures such as blood vessels in traditional open surgery. Over the past two decades, surgeons have shifted to ...minimally invasive surgical (MIS) approaches, including laparoscopic surgery, which include benefits such as less scarring, less risk for infection, and quicker recovery times. In fact, some surgeries such as cholecystectomies have seen more than an 80% adoption of this technique because of those benefits. However, due to the fundamental challenges associated with using laparoscopic surgery, there has been a lower adoption in more complex specialties, such as colorectal and thoracic surgery, where the field of surgery has bleeding, fat, scar tissue, and adhesions. These problems are exacerbated by complicating factors such as inflammation, cancer, chronic disease, obesity, and re-operations. Importantly, surgeons will often convert from laparoscopy to open surgery if they can no longer proceed using the minimally invasive approach because of issues described with these complicating factors, thereby negating the benefits that the patient would have seen. When the surgeon does attempt these procedures with those issues, the surgery takes on average 30 min - 1 hour longer. A new method by which surgeons can visualize structures like blood vessels could reduce the conversion rates and operating time, thereby driving a greater adoption of laparoscopic surgery in these complex procedures. Here, we show that by adding near infrared (NIR) LEDs and a linear image sensor onto the opposing jaws of the laparoscopic graspers, blood vessels that are embedded within tissues can be detected and localized efficiently, even those not visible using current imaging techniques. We show the results of Monte Carlo simulations to support our claim, including that blood vessels ranging from 2 to 6 mm and buried under up to 1 cm of tissue can be detected. We also report developing a smart grasper handheld prototype to run
experiments. The results of these experiments matched with those of the Monte Carlo simulations and the estimated blood vessel size showed a strong correlation with the actual size. This technology will be incorporated into already existing laparoscopic tools to assist surgeons during MIS procedures.
Abstract Background: Steroid hormones, such as progesterone, are known to have immunomodulatory effects. Our research group previously reported direct effects of progesterone on dendritic cells (DCs) ...from female rodents. Primarily affecting mature DC function, progesterone effects included inhibition of proinflammatory cytokine secretion, downregulation of cell surface marker (major histocompatibility complex class II, CD80) expression, and decreased T-cell proliferative capacity, and were likely mediated through progesterone receptor (PR) because the PR antagonist RU486 reversed these effects. Objective: The goal of this study was to assess differences in response to progesterone by DCs from female and male rodents. Methods: Using real-time reverse-transcriptase polymerase chain reaction, transcriptional expression of steroid hormone receptors was measured in immature bone marrow-derived DCs (BMDCs) from male and female rats. Expression of steroid hormone receptor protein was also assessed in these cells using flow cytometry and fluorescence microscopy. To evaluate functional differences between BMDCs from female and male rats in response to the steroid hormone progesterone, levels of secreted cytokines were measured using enzyme-linked immunosorbent assay. Results: Higher numbers of immature BMDCs from males expressed glucocorticoid receptor (GR) and androgen receptor (AR) proteins compared with females (males vs females, mean SD: GR = 68.75 7.27 vs 43.61 13.97, P = NS; AR = 75.99 15.38 vs 8.25 1.88, P = 0.002), whereas higher numbers of immature BMDCs from females expressed PR protein compared with males (females vs males: PR = 74.19 12.11 vs 14.14 4.55, P = 0.043). These differences were not found at the level of transcription (females vs males: GR = 0.088 vs 0.073, P = NS; AR = 0.076 vs 0.069, P = NS; PR = 0.075 vs 0.065, P = NS). Compared with those from females, mature BMDCs from males produced higher quantities of cytokines (tumor necrosis factor-α TNF-α, interleukin IL-1β, IL-10) (females vs males: TNF-α = 920.0 79.25 vs 1100.61 107.97, P = NS; IL-1β = 146.60 38.04 vs 191.10 10.47, P = NS; IL-10 = 167.25 4.50 vs 206.15 23.48, P = NS). Conversely, BMDCs from females were more sensitive to progesterone, as indicated by a more dramatic reduction in proinflammatory cytokine secretion (females vs males, highest concentration of progesterone: TNF-α = 268.94 28.59 vs 589.91 100.98, P = 0.04; IL-1β = 119.50 10.32 vs 154.35 6.22, P = NS). Conclusions: These findings suggest that progesterone effects on DCs in rodents may be more pronounced in females than in males, and this is likely due to differences in PR protein expression. Our observations may help elucidate disparities in the incidence and severity of autoimmune disorders between females and males, and the role specific steroid hormones play in regulating immune responses.
Cervical and vaginal epithelia are primary barriers against HIV type I (HIV-1) entry during male-to-female transmission. Cervical mucus (CM) is produced by the endocervix and forms a layer locally as ...well as in the vaginal compartment in the form of cervicovaginal mucus (CVM). To study the potential barrier function of each mucus type during HIV-1 transmission, we quantified HIV-1 mobility in CM and CVM ex vivo using fluorescent microscopy. Virions and 200-nm PEGylated beads were digitally tracked and mean-squared displacement was calculated. The mobility of beads increased significantly in CVM compared with CM, consistent with the known decreased mucin concentration of CVM. Unexpectedly, HIV-1 diffusion was significantly hindered in the same CVM samples in which bead diffusion was unhindered. Inhibition of virus transport was envelope-independent. Our results reveal a previously unknown activity in CVM that is capable of impeding HIV-1 mobility to enhance mucosal barrier function.
Several methods are currently employed to evaluate expression of steroid hormone receptors in tissues and cells, including real-time reverse-transcriptase polymerase chain reaction (real-time RT-PCR) ...and western blot assays. These methods require homogenization of cells, thereby preventing evaluation of individual cells or specific cell types in a given tissue sample. In addition, methods such as real-time RT-PCR assess mRNA levels, which may be subject to posttranslational modifications that prevent subsequent production of functional proteins. Flow cytometry is a fluorescence-based technique commonly used to evaluate expression of cell surface and intracellular proteins. This method is especially useful as it allows for single-cell analysis and can be utilized to determine the amount of receptor expressed by individual cells. Flow cytometry is commonly used to analyze immune cell activity and determine functionality based on changes in expression of cell surface molecules, as well as intracellular proteins (such as cytokines). Here, we describe a method to identify protein expression of steroid hormone receptors by rat leukocytes from different organs (spleen, liver and thymus) using flow cytometry. We examined expression of glucocorticoid receptor (GR), androgen receptor (AR) and progesterone receptor (PR) by cells at these sites and were able to demonstrate expression of receptors, as well as the intensity of expression of each receptor. This method is useful for rapid, high throughput measurement of steroid hormone receptors at the protein level in single, intact cells and would be valuable to determine which cells are more likely to respond to steroid hormone treatment.
Abstract In conjunction with the routine role of delivering the active ingredient, carefully designed drug delivery vehicles can also provide ancillary functions that augment the overall efficacy of ...the system. Inspired by the ability of the cervicovaginal mucus to impede the movement of HIV virions at acidic pH, we have engineered a pH-responsive synthetic polymer that shows improved barrier properties over the naturally occurring cervicovaginal mucus by inhibiting viral transport at both acidic and neutral pH. The pH-responsive synthetic mucin-like polymer is constructed with phenylboronic acid (PBA) and salicylhydroxamic acid (SHA), each individually copolymerized with a 2-hydroxypropyl methacrylamide (pHPMA) polymer backbone. At pH 4.8, the crosslinked polymers form a transient network with a characteristic relaxation time of 0.9 s and elastic modulus of 11 Pa. On addition of semen, the polymers form a densely crosslinked elastic network with a characteristic relaxation time greater than 60 s and elastic modulus of 1800 Pa. Interactions between the PBA-SHA crosslinked polymers and mucin at acidic pH showed a significant increase in elastic modulus and crosslink lifetime ( p < 0.05). A transport assay revealed that migration of HIV and cells was significantly impeded by the polymer network at pH ≥ 4.8 with a diffusion coefficient of 1.60 x 10−4 μm2 /s for HIV. Additionally, these crosslinked polymers did not induce symptoms of toxicity or irritation in either human vaginal explants or a mouse model. In summary, the pH-responsive crosslinked polymer system reported here holds promise as a class of microbicide delivery vehicle that could inhibit the transport of virions from semen to the target tissue and, thereby, contribute to the overall activity of the microbicide formulation.
PDF
