Search

Background: Human tissue/organ development is a complex, highly orchestrated process, regulated in part by the surrounding extracellular matrix (ECM). Every complex tissue, including the retina, has a unique ECM configuration that plays a critical role in cellular differentiation, adhesion, migration, and maturation. Aim: To characterize ECM expression of human induced pluripotent stem cell-derived optic vesicles (iPSC-OVs). Methods: A 3- dimensional (3D) in vitro suspension culture system was used to direct differentiation of human induced pluripotent stem cells (iPSCs) into optic vesicles (OVs). Stepwise differentiation of iPSCs into retinal progenitor cells was confirmed by sequential expression of OTX2, SOX1, SIX6, LHX2, PAX6, and CHX10. Expression of ECM genes in iPSC-derived OVs was analyzed by RT2 ProfilerTM PCR Array, whereas immunofluorescence staining was performed to detect ECM proteins in the OVs. Results: A number of cell adhesion molecules (CAMs) previously reported to be abundantly expressed in iPSCs such as E-cadherin, Intercellular adhesion molecule-1 (ICAM1), Integrin-α L, Integrin-α M, Integrin-α 6 were downregulated while neural and retina specific CAMs including neural cell adhesion molecule 1 (NCAM1), neural plakophilin-related armadillo repeat protein (NPRAP), Integrin-α 1 and Integrin-α 4 were upregulated. Several glycoproteins that have been reported to play key roles during retinogenesis, namely CD44, Tenascin C, Tenascin R, Neurocan, Neuroglycan C, Delta 2 Catenin, Vitronectin, and Reelin were also present. Conclusion: We have identified an array of ECM proteins that were expressed during retinogenesis. Further characterization of these proteins will lead to a better understanding of retinal development.

Traumatic brain injury (TBI) and hemorrhage remain challenging to treat in austere conditions. Developing a therapeutic to mitigate the associated pathophysiology is critical to meet this treatment gap, especially as these injuries and associated high mortality are possibly preventable. Here, Thera-101 (T-101) was evaluated as low-volume resuscitative fluid in a rat model of TBI and hemorrhage. The therapeutic, T-101, is uniquely situated as a TBI and hemorrhage intervention. It contains a cocktail of proteins and microvesicles from the secretome of adipose-derived mesenchymal stromal cells that can act on repair and regenerative mechanisms associated with poly-trauma. T-101 efficacy was determined at 4, 24, 48, and 72 h post-injury by evaluating blood chemistry, inflammatory chemo/cytokines, histology, and diffusion tensor imaging. Blood chemistry indicated that T-101 reduced the markers of liver damage to Sham levels while the levels remained elevated with the control (saline) resuscitative fluid. Histology supports the potential protective effects of T-101 on the kidneys. Diffusion tensor imaging showed that the injury caused the most damage to the corpus callosum and the fimbria. Immunohistochemistry suggests that T-101 may mitigate astrocyte activation at 72 h. Together, these data suggest that T-101 may serve as a potential field deployable low-volume resuscitation therapeutic.

OBJECTIVE Describe etiologies and trends in non-battle deaths (NBD) among deployed U.S. service members to identify areas for prevention. BACKGROUND Injuries in combat are categorized as battle (result of hostile action) or nonbattle related. Previous work found that one-third of injured US military personnel in Iraq and Afghanistan had nonbattle injuries and emphasized prevention. NBD have not yet been characterized. METHODS U.S. military casualty data for Iraq and Afghanistan from 2001 to 2014 were obtained from the Defense Casualty Analysis System (DCAS) and the Department of Defense Trauma Registry (DoDTR). Two databases were used because DoDTR does not capture prehospital deaths, while DCAS does not contain clinical details. Nonbattle injuries and NBD were identified, etiologies classified, and NBD trends were assessed using a weighted moving average and time-series analysis with autoregressive integrated moving average. Future NBD rates were forecast. RESULTS DCAS recorded 59,799 casualties; 21.0% (n = 1431) of all deaths (n = 6745) were NBD. DoDTR recorded 29,958 casualties; 11.5% (n = 206) of all deaths (n = 1788) were NBD. After early fluctuations, NBD rates for both Iraq and Afghanistan stabilized at approximately 21%. Leading causes of NBD were gunshot wounds and vehicle accidents, accounting for 66%. Approximately 25% was self-inflicted. A 24% NBD rate was forecasted from 2015 through 2025. CONCLUSIONS Approximately 1 in 5 deaths were NBD. The majority were potentially preventable, including a significant proportion of self-inflicted injuries. A single comprehensive data repository would facilitate future mortality monitoring and performance improvement. These data may assist military leaders with implementing targeted safety strategies.

Traumatic brain injury (TBI) and hemorrhage remain challenging to treat in austere conditions. Developing a therapeutic to mitigate the associated pathophysiology is critical to meet this treatment gap, especially as these injuries and associated high mortality are possibly preventable. Here, Thera-101 (T-101) was evaluated as an adjunct to low volume resuscitation in a rat model of TBI and hemorrhage. The therapeutic, T-101, is uniquely situated as a TBI and hemorrhage intervention. It contains a cocktail of proteins and microvesicles from the secretome of adipose derived mesenchymal stem cells that can act on numerous mechanisms associated with poly-trauma. T-101 efficacy was determined at four, 24, 48, and 72 hours after injury by evaluating blood chemistry, inflammatory chemo/cytokines, histology, and diffusion tensor imaging. Blood chemistry indicated that T-101 reduces markers of liver damage to Sham levels while the levels remain elevated with the vehicle control. Histology supports the potential protective effects of T-101 on the kidneys. Diffusion tensor imaging shows that the injury caused the most damage to the corpus callosum and the fimbria. Immunohistochemistry suggests that T-101 may mitigate astrocyte activation at 72 hours. Together, these data suggest T-101 could be a potential field deployable adjunct for low volume resuscitation.

Trauma-induced, critical-size bone defects pose a clinical challenge to heal. Albeit autografts are the standard-of-care, they are limited by their inability to be shaped to various defect geometries and often incur donor site complications. Herein, the combination of a "self-fitting" shape memory polymer (SMP) scaffold and seeded mesenchymal stromal cells (MSCs) was investigated as an alternative. The porous SMP scaffold, prepared from poly(ε-caprolactone) diacrylate (PCL-DA) and coated with polydopamine, provided conformal shaping and cell adhesion. MSCs from five tissues, amniotic (AMSCs), chorionic tissue (CHSCs), umbilical cord (UCSCs), adipose (ADSCs), and bone marrow (BMSCs) were evaluated for viability, density, and osteogenic differentiation on the SMP scaffold. BMSCs exhibited the fastest increase in cell density by day 3, but after day 10, CHSCs, UCSCs, and ADSCs approached similar cell density. BMSCs also showed the greatest calcification among the cell types, followed closely by ADSCs, CHSCs and AMSCs. Alkaline phosphatase (ALP) activity peaked at day 7 for AMSCs, UCSCs, ADSCs and BMSCs, and at day 14 for CHSCs, which had the greatest overall ALP activity. Of all the cell types, only scaffolds cultured with ADSCs in osteogenic media had increased hardness and local modulus as compared to blank scaffolds after 21 days of cell culture and osteogenic differentiation. Overall, ADSCs performed most favorably on the SMP scaffold. The SMP scaffold was able to support key cellular behaviors of MSCs and could potentially be a viable, regenerative alternative to autograft.

The advent of stem cell technology, including the technology to induce pluripotency in somatic cells, and direct differentiation of stem cells into specific somatic cell types, has created an exciting new field of scientific research. Much of the work with pluripotent stem (PS) cells has been focused on the exploration and exploitation of their potential as cells/tissue replacement therapies for personalized medicine. However, PS and stem cell-derived somatic cells are also proving to be valuable tools to study disease pathology and tissue-specific responses to injury. High-throughput drug screening assays using tissue-specific injury models have the potential to identify specific and effective treatments that will promote wound healing. Retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE) are well characterized cells that exhibit the phenotype and functions of in vivo RPE. In addition to their role as a source of cells to replace damaged or diseased RPE, iPS-RPE provide a robust platform for in vitro drug screening to identify novel therapeutics to promote healing and repair of ocular tissues after injury. Proliferative vitreoretinopathy (PVR) is an abnormal wound healing process that occurs after retinal tears or detachments. In this chapter, the role of iPS-RPE in the development of an in vitro model of PVR is described. Comprehensive analyses of the iPS-RPE response to injury suggests that these cells provide a physiologically relevant tool to investigate the cellular mechanisms of the three phases of PVR pathology: migration, proliferation, and contraction. This in vitro model will provide valuable information regarding cellular wound healing responses specific to RPE and enable the identification of effective therapeutics.

Accidental laser exposure to the eyes may result in serious visual impairment due to retina degeneration. Currently limited treatment is available for laser eye injury. In the current study, we investigated the therapeutic potential of bone marrow-derived stem cells (BMSCs) for laser-induced retinal trauma. Lineage negative bone marrow cells (Lin− BMCs) were labelled with quantum dots (Qdots) to track the cells in vivo. Lin− BMCs survived well after intravitreal injection. In vivo bromodeoxyuridine (BrdU) labelling showed these cells continued to proliferate and integrate into injured retinas. Furthermore, they expressed markers that distinguished retinal pigment epithelium (RPE), endothelium, pericytes and photoreceptors. Our results suggest that BMSCs participate in the repair of retinal lesions by differentiating into retinal cells. Intravitreal transplantation of BMSCs is a potential treatment for laser-induced retinal trauma.

Cryptosporidium parasites are pathogens of human intestinal epithelial cells. To determine which genes are regulated during early infection, human ileal mucosa cultured as explants was infected with C. parvum or C. hominis, and gene expression was analyzed by microarray. The gene for osteoprotegerin (OPG) was up-regulated by both parasites. OPG mRNA was also significantly increased in biopsy specimens obtained from a volunteer experimentally infected with C. meleagridis, compared with levels in a prechallenge biopsy specimen. After in vitro infection of HCT-8 cells, there was an early peak in production of OPG mRNA protein. Treatment of infected cells with the OPG ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced epithelial cell apoptosis and reduced parasite numbers, and recombinant OPG blocked these effects. These results suggest a novel TRAIL-mediated pathway for elimination of Cryptosporidium infection and a role for OPG in modulating this host response.

This study has examined the stimulatory and costimulatory effects of IL-18 on two subsets of murine small intestinal intraepithelial lymphocytes (IELs) defined by the expression of the CD43 S7 glycoform. Data from gene array studies and real-time PCR indicated that S7+ IELs had significantly higher levels of gene expression for the IL-18 receptor and the IL-18R accessory protein than S7− IELs. IL-18 costimulation of IELs in conjunction with CD3-induced activation resulted in significantly greater proliferation than CD3 stimulation alone. In CFSE dilution experiments, IL-18 costimulation favored the S7+ IEL population. IL-18 costimulation did not affect apoptosis of either S7− or S7+ IELs compared with CD3 stimulation alone. Although IL-18 costimulation did not alter the total number of IFN-γ-producing cells relative to CD3 stimulation alone, twice as many S7+ IELs were IFN-γ -secreting cells than S7− IELs in both CD3-stimulated and IL-18-costimulated cultures. Notably, direct IL-18 stimulation in the absence of CD3 activation induced an IFN-γ response that was predominantly directed to the S7+ population, indicating that IL-18 is itself an IFN-γ activational signal for intestinal T cells. In contrast, direct IL-18 stimulation of IELs did not generate TNF-α-producing cells, indicating a differential response in the activation of proinflammatory cytokines following IL-18 exposure. These findings point to distinctly different activational effects of IL-18 on IELs, both with regard to the type of functional responses elicited and with respect to the IEL subsets affected.

SUMMARYImmunofluorescent staining of thyroid tissues was done using monoclonal antibodies to dendritic cell (DC), lymphocyte, macrophage and granulocyte markers. Despite the presence of occasional CD11c+ cells,CD11b+ cells, morphologically characteristic of DCs, were abundant in thyroid of normal mice, at a density of ∼2.0 cells per thyroid follicle, and were >tenfold more frequent than CD11c+ cells. Thyroid tissues were non-reactive with antibodies to F4/80, CD8α, CD40,CD80, Gr-1, CD3, or CD19, indicating that the CD11b+ cells were not macrophages, activated DCs, granulocytes, plasmacytoid DCs, T cells or B cells. Following systemic immune activation, DCs in secondary lymphoid tissues but not in the thyroid, upregulated CD80 expression. Using radiation chimeras made from bone marrow from enhanced green fluorescent protein (EGFP)transgenic mice, EGFP+ DC-like cells were present in the thyroid from 1–20 weeks after bone marrow transfer, but were rare in the kidney and liver, although EGFP+ cells were present in secondary lymphoid tissues. Additionally, DCs generated from EGFP+ bone marrow cells localized in the thyroid of EGFP– mice following adoptive transfer. Double staining of thyroid tissue sections with antibodies to the thyroid stimulating hormone (TSH)-β molecule and to CD11b revealed co-expression of TSHβ and CD11b among intrathyroidal DCs. Moreover,RT-PCR analyses indicated expression of the TSHβ gene in thyroid tissues. These findings define a novel bone marrow-derived hematopoietic cell population that resides in the thyroid of normal mice, which may have a unique role in the microregulation of thyroid physiology and homeostasis.

Recent studies have identified a role for thyroid-stimulating hormone (TSH; ie, thyrotropin) as an inductive signal for tumor necrosis factor-α (TNF-α) secretion by bone marrow (BM) cells, although the features of that activation pathway have not been defined. Using intracellular TSH staining and enzyme-linked immunoassay for detection of secreted TSH, we demonstrate that TSH synthesis in BM cells occurs within CD45+ (leukocyte common antigen) hematopoietic cells and that the majority of that activity resides in a component of CD11b+ BM cells that are not mature T cells, B cells, or Thy-1+ cells in the BM. Conversely, TSH-responsive BM cells defined by expression of TSH receptor (TSHR) using flow cytometry were selectively associated with a nonerythroid CD11b− lymphocyte precursor population. In vitro culture of magnetic-activated cell sorted CD11b− and CD11b+ cells with titrated amounts of purified TSH resulted in significantly higher levels of TNF-α secretion from CD11b− BM cells compared to non-TSH–treated cells, with no appreciable change in TNF-α production from CD11b+cells. These findings are the first to demonstrate TSH production by BM hematopoietic cells, and they demonstrate that TSH may be involved in the regulation of TNF-α by CD11b− BM cells. They also indicate that TSH-mediated regulation of TNF-α secretion within the BM most likely operates through an intrinsic network of TSH production and use between different types of BM cells, and they suggest that local TSH may be an important homeostatic regulator of hematopoiesis mediated by TNF-α.

Murine small intestine intraepithelial lymphocytes (IELs) bear properties of both activated and nonactivated T cells, although the significance of that dichotomy remains unclear. In this study, we show that although IELs express CD69 in situ and ex vivo, and have cytotoxic activity ex vivo, most CD8+ IELs from normal mice are phenotypically similar to naive T cells in that they are CD45RBhigh, CD44low/int, and lack or have low levels of expression of CD25, Ly-6C, OX40, Fas ligand (FasL), and intracellular IFN-γ synthesis. Unlike CD8+ lymph node cells, IELs express high levels of the FasL gene, but do not express surface FasL until after CD3-mediated stimulation has occurred. Additionally, anti-CD3 stimulation of IELs in the presence of actinomycin-D did not inhibit FasL expression, suggesting that regulation FasL expression on IELs is controlled at least partially at the posttranscriptional level. Following CD3-mediated stimulation, IELs synthesize and secrete IFN-γ more rapidly and to greater levels than CD8+ lymph node cells, and they acquire the phenotype of fully activated effector cells as seen by an up-regulation of CD44, Ly-6C, OX40, FasL, and CD25 with the kinetics of memory T cells, with down-regulation of CD45RB expression. These findings indicate that contrary to previous interpretations, most small intestine IELs are not fully activated T cells, but rather that they are semiactivated T cells ready to shift to a fully activated state once a CD3-mediated signal has been received. These data also imply that under appropriate conditions it is possible for T cells to be sustained in a state of partial activation.

T cells using the γδ T cell receptor (TCR) are abundant in mucosal and epidermal tissues in mice. Most studies of mucosal γδ T cells, however, have examined cells from the intestinal mucosa, whereas little is known about the presence or function of γδ T cells in the oral cavity. To better understand the involvement of oral γδ T cells in immunity, we have characterized TCR variable γ-gene usage in the buccal epithelium from normal mice, and from mice challenged locally with a non-replicating antigen (bovine serum albumin [BSA]) or by influenza-virus infection as a replicating antigen. Our findings demonstrate a restricted use of Vγ genes by buccal γδ T cells, consisting primarily of Vγ1.2, Vγ3, and Vγ5, with minimal use of Vγ2 and Vγ4 genes. Of particular interest, 3–4 days post-antigen challenge with BSA, there was a precipitous drop in the level of expression of Vγ1.2, Vγ3, and Vγ5 genes, and to a lesser extent for the Vγ2 gene, whereas Vγ4 gene expression increased between days 1 and 2 post-priming. In influenza-infected mice, a similar pattern was observed for the Vγ2 and Vγ5 genes, but not other Vγ genes. The immune-modulating effects of oral antigen exposure on buccal γδ T cells suggest that these cells are functionally involved in the local immune response to both replicating and non-replicating antigens in oral mucosal surfaces.

The incidence of blast-induced ocular injury has dramatically increased due to advances in weaponry and military tactics. A single exposure to blast overpressure (BOP) has been shown to cause damage to the eye in animal models; however, on the battlefield, military personnel are exposed to BOP multiple times. The effects of repeated exposures to BOP on ocular tissues have not been investigated. The purpose of this study is to characterize the effects of single or repeated exposure on ocular tissues.A compressed air shock tube was used to deliver 70 ± 7 KPa BOP to rats, once (single blast overpressure [SBOP]) or once daily for 5 days (repeated blast overpressure [RBOP]). Immunohistochemistry was performed to characterize the pathophysiology of ocular injuries induced by SBOP and RBOP. Apoptosis was determined by quantification activated caspase 3. Gliosis was examined by detection of glial fibrillary acidic protein (GFAP). Inflammation was examined by detection of CD68.Activated caspase 3 was detected in ocular tissues from all animals subjected to BOP, while those exposed to RBOP had more activated caspase 3 in the optic nerve than those exposed to SBOP. GFAP was detected in the retinas from all animals subjected to BOP. CD68 was detected in optic nerves from all animals exposed to BOP.SBOP and RBOP induced retinal damage. RBOP caused more apoptosis in the optic nerve than SBOP, suggesting that RBOP causes more severe optic neuropathy than SBOP. SBOP and RBOP caused gliosis in the retina and increased inflammation in the optic nerve.

The involvement of OX40 (CD134) in the activation of CD8+ intestinal intraepithelial lymphocytes (IELs) has been studied using freshly isolated IELs and in vitro CD3-stimulated IELs. Although freshly isolated CD8+ IELs exhibited properties of activated T cells (CD69 expression and ex vivo cytotoxicity), virtually all CD8+ IELs from normal mice were devoid of other activation-associated properties, including a lack of expression of OX40 and the ligand for OX40 (OX40L) and an absence of intracellular IFN-γ staining. However, OX40 and OX40L expression were rapidly up-regulated on CD8 IELs following CD3 stimulation, indicating that both markers on IELs reflect activation-dependent events. Unlike IELs, activated lymph node T cells did not express OX40L, thus indicating that OX40-OX40L communication in the intestinal epithelium is part of a novel CD8 network. Functionally, OX40 expression was exclusively associated with IELs with active intracellular IFN-γ synthesis and markedly enhanced cell-mediated cytotoxicity. However, OX40 costimulation during CD3-mediated activation significantly suppressed IL-10 synthesis by IELs, whereas blockade of OX40-OX40L by anti-OX40L mAb markedly increased IL-10 production. These findings indicate that: 1) resident CD69+OX40− IELs constitute a population of partially activated T cells poised for rapid delivery of effector activity, 2) OX40 and OX40L expression defines IELs that have undergone recent immune activation, 3) OX40+ IELs are significantly more efficient CTL than are OX40− IELs, and 4) the local OX40/OX40L system plays a critical role in regulating the magnitude of cytokine responses in the gut epithelium.

The thyroid hormones T(3) (tri-iodothyronine) and T(4) (thyroxine) are disseminated throughout the body via the circulation and are maintained across a range of physiological concentrations under the control of thyroid-stimulating hormone (TSH). T(3) (and T(4) after conversion to T(3)) influences many biological activities, including gene expression and protein synthesis, though little is known about the nature of pituitary-thyroid immune interactions. In the present study we show that serum T(3) and T(4) levels are sharply but transiently reduced during the first 24 h of systemic antigen exposure and that this is followed by suppressed levels of free T(4), after which there is rapid recovery to normal levels. Splenic dendritic cells, depending upon the stage of maturation/activation, were found to be a rich source of TSH, and CD11c(+) cells with dendritic cell morphology were present in the thyroid 1-3 days after antigen exposure. Moreover, antigen priming of hypophysectomized mice that are unable to make pituitary-derived TSH resulted in significant increases in circulating T(4), implying that compensation in the drop in thyroid hormones can be regulated from extrapituitary sources. These findings thus identify a novel set of immune-endocrine interactions that transpire during the early phase of antigen exposure, and they suggest that under appropriate conditions the immune system directly participates in the process of maintaining physiological homeostasis by contributing to the regulatory control of thyroid hormone activity.

The involvement of CD43 in cell proliferation of murine intestinal intraepithelial lymphocytes (IEL) has been studied in in vitro CD3-stimulated cell cultures. In the presence of either IL-2 or IL-15, CD3 stimulation of IEL resulted in low levels of proliferation as measured by thymidine incorporation, whereas no proliferation occurred upon CD3 stimulation in the absence of cytokines. The combination of both cytokines to IEL cultures synergistically enhanced CD3-induced proliferation by approximately threefold that of cultures supplemented with either cytokine alone. Most importantly, however, proliferation of IEL was significantly greater when CD3 stimulation occurred in conjunction with CD43 triggering, indicating that CD43 functions as a coactivational signal for murine IEL. These findings indicate that a spectrum of potential proliferative responses exist among murine IEL depending on the types and combinations of signals received, and that because under normal conditions murine IEL are largely devoid of CD28 expression, a classical T-cell coactivational molecule, the capacity for high-level IEL proliferation may reside with CD43.

Cryptosporidiosis is an important cause of diarrhea worldwide. In normal hosts, infection is self-limited and associated with seroconversion and partial immunity to reinfection. Immunity is associated with interferon gamma (IFN) production. Cryptosporidium surface proteins gp15 and gp40 are among the immunodominant proteins in terms of antibody responses. We asked the question of whether these antigens also stimulate production of IFN in patients who have serologic evidence of prior infection. Whole blood from seropositive donors was stimulated with recombinant gp15 and gp 40 from Cryptosporidium hominis and Cryptosporidium parvum or His-tag controls. C. hominis gp15 stimulated increased production of IFN. By contrast, there was no significant increase after stimulation with C. parvum gp15 or either gp40 preparation. IFN production in response to C. hominis gp15 was noted in both CD4 + and CD8 + cells. This highlights the potential for C. hominis gp15 as a vaccine candidate for human cryptosporidiosis. Cryptosporidiosis is an important cause of diarrhea world- wide. 1 The host immune response controls the disease in im- munocompetent patients, and recovery is associated with re- sistance to reinfection. By contrast, cryptosporidiosis can be chronic and fatal in AIDS patients and malnourished chil- dren. Cytokines, particularly interferon gamma (IFN), are critical in the immune response controlling cryptosporidiosis. IFN-knockout mice develop chronic, severe infections. 2 Lymphocytes from people who have recovered from cryptosporidiosis produce IFN after antigen stimulation in vitro. 3,4 In volunteer studies, expression of IFN was associ- ated with resistance to infection and prior sensitization. 5 Thus, expression of IFN is an important marker of immunity to infection.

The spatial and temporal activation of NF-kB (p65) was monitored in the retina of a transgenic mouse model (cis-NFkB-EGFP) in vivo after receiving varying grades of laser induced thermal injury in one eye. Baseline images of the retinas from 26 mice were collected prior to injury and up to five months post-exposure using a Heidelberg Spectralis HRA confocal scanning laser ophthalmoscope (cSLO) with a spectral domain optical coherence tomographer (SDOCT). Injured and control eyes were enucleated at discrete time points following laser exposure for cryosectioning to determine localization of NF-kB dependent enhanced green fluorescent protein (EGFP) reporter gene expression within the retina using fluorescence microscopy. In addition, EGFP basal expression in brain and retinal tissue from the cis-NFkB-EGFP was characterized using two-photon imaging. Regions of the retina exposed to threshold and supra-threshold laser damage evaluated using fluorescence cSLO showed increased EGFP fluorescence localized to the exposed region for a duration that was dependent upon the degree of injury. Fluorescence microscopy of threshold damage revealed EGFP localized to the outer nuclear region and retinal pigment epithelial layer. Basal expression of EGFP imaged using two-photon microscopy was heterogeneously distributed throughout brain tissue and confined to the inner retina. Results show cis-NF-kB-EGFP reporter mouse can be used for in vivo studies of light induced injury to the retina and possibly brain injury.

Cryptosporidium is an obligate intracellular protozoan parasite that is a major cause of diarrheal illness worldwide. Cryptosporidium primarily infects the distal small intestine. Immunocompetent hosts control and eliminate the infection, which typically causes acute, self-limited watery diarrhea lasting 5 to 10 days. However, in patients with defects in cellular immune responses (e.g., AIDS, malnutrition, or defects in the CD40-CD154 system), Cryptosporidium frequently causes persistent or chronic diarrhea and may also involve the biliary tract (40). In malnourished children, persistent diarrhea is associated with increased susceptibility to recurrent diarrheal episodes, which can lead to death or chronic nutritional and cognitive sequelae (1, 9, 33). Thus, the host immune response plays a critical role in the control of human cryptosporidiosis. Although extensive studies with various animal models have provided important insight into the host immune response towards Cryptosporidium parvum, the ability of these models to explain the human immune response is limited. The clinical picture in rodents differs from that in humans, as mice do not get diarrhea after infection. Nonhuman primates, although probably the best in vivo model to mimic human disease, are difficult to work with, expensive, and not widely available. Cryptosporidium hominis, the pathogen causing most human cryptosporidiosis, infects only humans and gnotobiotic pigs, thus limiting data from animal models. Most importantly, comparison of animal and human data has shown that the immune response towards Cryptosporidium in humans differs significantly from that in animals; for example, in mice gamma interferon (IFN-γ) production seems to be associated with the innate and primary immune responses (35, 47), whereas in humans it is most probably associated with the memory response towards the parasite (93). Conducting studies to elucidate human mucosal immune responses is difficult. Patients with a natural infection would be the ideal subjects to study, but it is difficult to identify cases. Healthy human volunteers can be studied, but they typically experience a milder illness than malnourished children and AIDS patients. Human intestinal tissue samples can be obtained only by invasive procedures, limiting the numbers of subjects and samples available. Some data can be obtained from in vitro infections, but most of the target cells are immortalized and may not be ideal for studying mechanisms involving apoptosis. Furthermore, the immune cells in the peripheral blood may exhibit properties different from the properties of cells found in the intestinal compartment. Thus, knowledge about the human immune response towards Cryptosporidium infection is far from complete. Still, important recent advances have been made. The goal of this paper is to review the current literature to provide an understanding of the human immune response towards the parasite. We include some relevant data from other models only when the data shed light on studies performed with human cells or tissues.

Cryptosporidiosis is an important cause of diarrhea worldwide. In normal hosts, infection is self-limited and associated with seroconversion and partial immunity to reinfection. Immunity is associated with interferon gamma (IFNgamma) production. Cryptosporidium surface proteins gp15 and gp40 are among the immunodominant proteins in terms of antibody responses. We asked the question of whether these antigens also stimulate production of IFNgamma in patients who have serologic evidence of prior infection. Whole blood from seropositive donors was stimulated with recombinant gp15 and gp 40 from Cryptosporidium hominis and Cryptosporidium parvum or His-tag controls. C. hominis gp15 stimulated increased production of IFNgamma. By contrast, there was no significant increase after stimulation with C. parvum gp15 or either gp40 preparation. IFNgamma production in response to C. hominis gp15 was noted in both CD4(+) and CD8(+) cells. This highlights the potential for C. hominis gp15 as a vaccine candidate for human cryptosporidiosis.

Chemokines play key roles in attracting immune cells to sites of infections. However, few data on chemokine expression in the gut during human infections are available. We examined expression of chemokines in intestinal tissues of AIDS patients during active Cryptosporidium infection and during resolution of such an infection. The chemokines and cytokines in cell lysates from jejunal biopsy tissues were assayed by a 22-multiplex bead immunoassay. CXCL10 (IP-10) and its receptor, CXCR3, in sections were studied by immunohistochemistry. In biopsies from AIDS patients with active cryptosporidiosis, four chemokines (CXCL10, CCL11 [eotaxin], CCL5 [RANTES], and CCL2 [monocyte chemoattractant protein 1]) and three cytokines (interleukin-1α [IL-1α], IL-10, and granulocyte colony-stimulating factor) were detected. The level of CXCL10 was significantly increased in AIDS patients with cryptosporidiosis compared to the level in AIDS patients without cryptosporidiosis or in normal volunteers (median in AIDS patients with cryptosporidiosis, 508 pg/mg protein, compared to 111 pg/mg and 72 pg/mg protein in AIDS patients without cryptosporidiosis and in normal volunteers, respectively [ P < 0.05 and P < 0.005, respectively, as determined by a Mann-Whitney test]). The level of CXCL10 correlated with the parasite burden (as measured by the number of Cryptosporidium oocysts in the stools) and also with the IL-1α concentration (Pearson correlation values, 0.961 [ P < 0.01] and 0.737 [ P < 0.05]). As determined by immunohistochemistry, CXCL10 localized to epithelial cells at the site of infection. Following effective antiparasite and antiretroviral therapy, Cryptosporidium infections resolved, and the levels of CXCL10 decreased to normal levels. We hypothesized that CXCL10 plays an important role in the resolution of cryptosporidiosis by attracting immune effector cells to the site of infection. By contrast, in AIDS patients lacking effector cells, CXCL10 may contribute to the immunopathogenesis by recruiting inflammatory cells.

Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, but little is known about the mechanisms that control intestinal infection. We have previously demonstrated interleukin (IL)-15 expression in the intestinal mucosa of seronegative symptomatic volunteers after oral challenge with C. parvum, which suggests a role for IL-15 in the control of acute infection. We hypothesize that IL-15 activates an innate cytolytic cell response that contributes to the clearance of initial C. parvum infection. We report here that IL-15 activates peripheral blood mononuclear cells to lyse Cryptosporidiuminfected epithelial cells in a dose-dependent manner. Lysis was due to CD3 - CD16 + CD56 + cells (i.e., natural killer [NK] cells). Furthermore, flow cytometry revealed that IL-15 increased expression of the activation receptor NKG2D on NK cells, particularly among the CD16 Hi cytolytically active cells. Major histocompatibility complex class I-related molecules A and B (MICA and MICB), ligands for NKG2D, were increased after infection of epithelial cell lines and human ileal tissue. These data suggest that IL-15 has an important role in activating an NK cell-mediated pathway that leads to the elimination of intracellular protozoans from the intestines, which is a previously unrecognized feature of NK cell function.

CD43 has been linked to many function-associated T cell activities. Using mAbs that recognize two different CD43 determinants, we show that, although mouse small intestinal intraepithelial lymphocytes (IELs) expressed the CD43 core molecule reactive with mAb R2/60, only about one-half of the total IELs—including some but not all of the TCRαβ and TCRγδ cells—expressed the CD43 S7− reactive determinant. CD43 S7+ IELs secreted more IL-2, IL-4, IL-10, IL-17, and IFN-γ following anti-CD3 stimulation, and were >4-fold more cytotoxic in fresh isolates and >16-fold more cytotoxic after anti-CD3 stimulation, than S7− IELs. S7+ but not S7− IELs from the ileum of IL-10−/− mice spontaneously produced IFN-γ. In vivo BrdU uptake by IELs in non-Ag-primed mice was greatest in the S7+ population, indicating that significantly more S7+ IELs than S7− IELs undergo cell expansion under normal homeostatic conditions. DNA microarray analyses showed that S7+ IELs expressed higher levels of genes associated with activated T cells, whereas S7− IELs expressed genes used in the regulation of NK cells. These findings define two functionally distinct populations of IELs based on CD43 expression independent of TCR class, and they identify a subset of IELs that may serve as a target to better control intestinal inflammation.

The oral mucosa is an active though poorly understood immunological site. Using an experimental animal system involving antigen priming into the oral mucosa of transgenic mice expressing T cell receptor (TCR) for a peptide antigen of hen-egg lysozyme (HEL), the expression of six chemokine receptors and seven chemokine ligands were studied before and after antigen exposure. Within 24h of local antigen priming, the expression of three chemokine receptor genes (CCR3, CCR5, and CCR7) and three chemokine ligand genes (CCL12, CCL19, and CCL25) were significantly upregulated. These included chemokines known to be responsible for the trafficking of T cells and other leukocytes into tissue sites. Additionally, expression of the chemokine ligand gene, CCL25 (thymus-expressed chemokine [TECK]), which has been linked to T cell migration and/or local T cell development in the intestine, was also markedly elevated in buccal epithelia after antigen exposure. These findings define a process of selective activation of proinflammatory chemokines and/or their receptors following local antigen exposure, and they provide the first evidence, indicating that this may be accompanied by in situ development of T cells in oral tissues.

T cells using the gamma delta T cell receptor (TCR) are abundant in mucosal and epidermal tissues in mice. Most studies of mucosal gamma delta T cells, however, have examined cells from the intestinal mucosa, whereas little is known about the presence or function of gamma delta T cells in the oral cavity. To better understand the involvement of oral gamma delta T cells in immunity, we have characterized TCR variable gamma-gene usage in the buccal epithelium from normal mice, and from mice challenged locally with a non-replicating antigen (bovine serum albumin [BSA]) or by influenza-virus infection as a replicating antigen. Our findings demonstrate a restricted use of V gamma genes by buccal gamma delta T cells, consisting primarily of V gamma 1.2, V gamma 3, and V gamma 5, with minimal use of V gamma 2 and V gamma 4 genes. Of particular interest, 3-4 days post-antigen challenge with BSA, there was a precipitous drop in the level of expression of V gamma 1.2, V gamma 3, and V gamma 5 genes, and to a lesser extent for the V gamma 2 gene, whereas V gamma 4 gene expression increased between days 1 and 2 post-priming. In influenza-infected mice, a similar pattern was observed for the V gamma 2 and V gamma 5 genes, but not other V gamma genes. The immune-modulating effects of oral antigen exposure on buccal gamma delta T cells suggest that these cells are functionally involved in the local immune response to both replicating and non-replicating antigens in oral mucosal surfaces.

Mouse hybridoma cell lines have been raised to a peptide of the mouse thyrotropin-beta (thyroid stimulating hormone [TSH]beta) subunit of the TSH glycoprotein hormone molecule. Using an enzyme-linked immunosorbent assay (ELISA) capture technique with two anti-TSHbeta monoclonal antibodies (MAbs), these reagents were found to have strong reactivity in a titration-dependent manner against normal mouse serum, and to precipitate under reducing conditions a 13-kDa product, the correct molecular size of the TSHbeta component, from mouse sera. These MAbs had minimal reactivity in ELISA to bovine and rat TSH and slight reactivity to human TSH, demonstrating overall strong species specificity for mouse TSH. Due to the large number of inbred and genetically manipulated mice now available for experimental research, it is anticipated that these reagents will be of considerable value for in-depth studies into TSH-related physiological processes in ways that have not been feasible here-to-fore.

Thyroid stimulating hormone (TSH) is a central component of the hypothalamus-pituitary-thyroid axis. Although TSH is known for its important biological effects as a neuroendocrine used to regulate thyroid hormone activity and subsequent metabolic functions, TSH also has been shown to be produced and used by cells ofthe mammalian immune system. Moreover, recent findings have linked the use of TSH by cells of the immune system in humans and mice to a group of monocytic cells and lymphocytes--primarily dendritic cells, macrophages, and subset of naive peripheral T cells. Other studies have demonstrated the capacity of dendritic cells and monocytes to produce biologically active TSH, thereby pointing to a process of paracrine or autocrine TSH-mediated communication during the earliest stages of an immune response to antigen. In this article, these and other features of TSH immune-endocrine interactions are discussed in the context of an intrinsic TSH immunological pathway. Additionally, a hypothesis is proposed in which TSH produced by cells of the immune system during acute antigen exposure plays a dual role, consisting on the one hand of TSH communication during antigen-driven immune activation while concomitantly serving to regulate physiological homeostasis by modulating and adjusting thyroid hormone activity.