Cells In Target Tissues Research Articles

Novel mechanism of rapamycin in GVHD: increase in interstitial regulatory T cells

Rapamycin (RAPA) is an immunosuppressive drug that prevents and treats graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplant (HCT). One possible mechanism for its efficacy is induction of tolerance, through increased number or enhanced survival of regulatory T cells. In our experiments, B10.D2 BM and splenocytes were injected into lethally irradiated BALB/cJ recipients. The mice received i.p. injections of either RAPA or vehicle control on days 1-28. There was a significant survival advantage in RAPA-treated mice. Evaluation of the skin biopsies showed a dense cellular infiltrate in RAPA-treated mice. Further characterization of these cells revealed a higher percentage of regulatory T cells characterized by FoxP3-positive cells in high-dose RAPA-treated mice as compared with controls on day 30. This effect appears to be dose dependent. When peripheral blood analysis for FoxP3-positive cells was performed, there was no significant difference observed in the RAPA-treated mice as compared with control mice. These data show a novel mechanism of rapamycin in GVHD, accumulation of regulatory T cells in the GVHD target tissue: the skin.

Vascularization—The Conduit to Viable Engineered Tissues

Long-term viability of thick three-dimensional engineered tissue constructs is a major challenge. Addressing it requires development of vessel-like network that will allow the survival of the construct in vitro and its integration in vivo owing to improved vascularization after implantation. Resulting from work of various research groups, several approaches were developed aiming engineered tissue vascularization: (1) embodiment of angiogenesis growth factors in the polymeric scaffolds for prolonged release, (2) coculture of endothelial cells with target tissue cells and angiogenesis signaling cells, (3) use of microfabrication methods for creating designed channels for allowing nutrients to flow and/or for directing endothelial cells attachment, and (4) decellularization of organs and blood vessels for creating extracellular matrix. A synergistic effect is expected by combining several of these approaches as already demonstrated in some of the latest studies. Current paper reviews the progress in each approach and recent achievements toward vascularization of engineered tissues.

IONTOPHORESIS-ENHANCED CUTANEOUS ABSORPTION OF 5-AMINOLEVULINIC ACID SHORTENS THE INCUBATION PERIOD IN PHOTODYNAMIC THERAPY

The combination of light and chemicals to treat skin diseases is widely practiced in the field of dermatology, and has led to the concept of photodynamic therapy (PDT) in recent years. PDT is a promising modality for managing various cancers and nonmalignant diseases, and is based on the combination of selective localization of a photosensitizer in the target tissue cells and the subsequent illumination of the lesions with visible light resulting in photodamage and cell death. PDT with topical 5-aminolevulinic acid (ALA) has been increasingly employed to treat skin cancers,however the dosing of ALA has never been optimized. Using iontophoresis, we have developed a rapid and quantifiable system for the topical delivery of ALA, and we measured the subsequent fluorescence of protoporphyrin IX (PpIX) and phototoxicity. Iontophoresis is a rapid method for the delivery of ALA into skin lesions, making prolonged incubation of the ALA unnecessary and potentially leading to more efficient PDT.

Radiation-Free Anti-CD3-Conditioning Regimen Maintains Tissue Protection Mechanisms and Prevents GVHD: Role of Tissue Expression of B7H1

We reported that donor CD8+ T cells mediated graft versus leukemia (GVL) activity without causing graft versus host disease (GVHD) in anti-CD3-conditioned recipients, although the same dose of donor CD8+ T cells caused lethal GVHD in recipients conditioned with sublethal total body irradiation (TBI) (J. Immunol. 2007). We recently observed that donor CD8+ T cells from the liver of anti-CD3-conditioned recipients showed a marked reduction in proliferation in response to anti-CD3/CD28 stimulation, as compared to those from TBI-conditioned recipients, indicating that donor CD8+ T cells are tolerized in the tissues of anti-CD3-conditioned recipients. B7H1, a co-inhibitory molecule, is constitutively expressed by hematopoietic cells and expressed by parenchymal cells after IFN-γ induction. B7H1 tolerizes T cells by interaction with its ligand PD-1 on activated T cells. To explore the role of B7H1 in GVHD prevention in anti-CD3-conditioned recipients, donor C57BL/6 CD8+ T cells (20×106) and bone marrow cells (100×106) were transplanted into anti-CD3-conditioned wild-type or B7H1−/− BALB/c recipients. While the wild-type recipients all survived for more than 100 days without signs of GVHD, the B7H1−/− recipients developed severe GVHD with diarrhea, weight-loss, and hair-loss, and 70% of them died 60 days after transplantation. Similarly, while donor CD8+ T and BM cells induced little GVHD in unconditioned Rag-2−/− BALB/c recipients, they induced severe lethal GVHD in B7H1−/− Rag-2−/− BALB/c recipients. Furthermore, donor CD8+ T and BM cells still induced lethal GVHD in unconditioned chimeric B7H1−/−Rag-2−/− recipients reconstituted with B7H1+/+Rag-2−/− BM. In addition, we observed upregulation of B7H1 expression by hepatocytes and intestine epithelial cells of anti-CD3-conditioned BALB/c or unconditioned Rag-2−/− recipients after donor cells infusion, as judged by RT-PCR, flow cytometry analysis, and histoimmunostaining of B7H1. In vivo bioluminescent imagine showed much more severe tissue infiltration of donor T cells in B7H1−/− recipients as compared to B7H1+/+ recipients, and the in vitro proliferation of donor CD8+ T cells from the liver of the former recipients was much more vigorous than that of the latter recipients. These results demonstrate that B7H1 expression by tissue parenchymal cells rather than hematopoietic cells tolerizes infiltrating donor T cells in GVHD target tissues and prevents GVHD; and that the radiation-free anti-CD3-conditioning regimen can maintain this tissue protection mechanism.(This study is surpported by Marcus Foundation and NIH R01 AI 066008).

In vivo–activated CD103+CD4+ regulatory T cells ameliorate ongoing chronic graft-versus-host disease

AbstractCD103 (αEβ7) has been shown to be an excellent marker for identifying in vivo–activated FoxP3+CD4+ regulatory T (Treg) cells. It is unknown whether reinfusion of in vivo–activated donor-type CD103+ Treg cells from recipient can ameliorate ongoing chronic graft-versus-host disease (GVHD). Here, we showed that, in a chronic GVHD model of DBA/2 (H-2d) donor to BALB/c (H-2d) recipient, donor-type CD103+ Treg cells from recipients were much more potent than CD25hi natural Treg cells from donors in reversing clinical signs of GVHD and tissue damage. Furthermore, in contrast to CD25hi natural Treg cells, CD103+ Treg cells expressed high levels of CCR5 but low levels of CD62L and directly migrated to GVHD target tissues. In addition, the CD103+ Treg cells strongly suppressed donor CD4+ T-cell proliferation; they also induced apoptosis of in vivo–activated CD4+ T and B cells and significantly reduced pathogenic T and B cells in GVHD target tissues. These results indicate that CD103+ Treg cells from chronic GVHD recipients are functional, and reinfusion of the CD103+ Treg cells can shift the balance between Treg cells and pathogenic T cells in chronic GVHD recipients and ameliorate ongoing disease.

Therapeutic potential of non-adherent BM-derived mesenchymal stem cells in tissue regeneration.

We demonstrated that non-adherent BM cells (NA-BMCs) can be expanded in suspension and give rise to multiple mesenchymal phenotypes including fibroblastic, osteoblastic, chondrocytic and adipocytic as well as glial cell lineages in vitro using the 'pour-off' BMC culture method. Mesenchymal stem cells (MSCs) derived from NA-BMCs (NA-MSCs) from wild-type mice were transplanted into VDR gene knockout (VDR(-/-)) mice that had received a lethal dose of radiation. Results revealed that NA-MSC can be used to rescue lethally irradiated mice and become incorporated into a diverse range of tissues. After lethal dose irradiation, all untransplanted mice died within 2 weeks, whereas those transplanted with NA-MSCs were viable for at least 3 months. Transplantation rescued these mice by reconstructing a hematopoietic system and repairing other damaged tissues. WBC, RBC and platelet counts recovered to normal after 1 month, and VDR gene expression was found in various tissues of viable VDR(-/-) recipients. Adult BM harbors pluripotent NA-MSCs, which can migrate in vivo into multiple body organs. In an appropriate microenvironment, they can adhere, proliferate and differentiate into specialized cells of target tissues and thus function in damaged tissue regeneration and repair.

TH17 Cells Mediate Extensive Skin Pathology in Acute GVHD.

INTRODUCTION: Cytokines play an integral role in the development of acute graft-versus-host disease (GVHD). In particular, IFN-gamma has been ascribed both pathologic as well as protective roles in murine models of GVHD. IFN-gamma is the trademark cytokine produced by differentiated T helper (TH) type 1 CD4+ T cells, whereas IL-4 is a characteristic cytokine produced by TH2 cells. Although a number of studies have investigated the role each of these cytokines have in development or inhibition of GVHD, little evidence has been gathered regarding the role of IL-17 producing T cells (TH17). TH17 cells have been attributed to mediating pathology in several autoimmune conditions that once were thought to be uncontrolled TH1 responses. IL-17 is a proinflammatory cytokine secreted exclusively by T cells that induces production of other proinflammatory cytokines by epithelial cells, macrophages, endothelial cells and fibroblasts. Elevated levels of IL-17 have been observed in rheumatoid arthritis, multiple sclerosis, and inflammatory bowl disease.METHODS: Given the highly proinflammatory properties of IL-17 we sought to examine the role of TH17s in acute GVHD. Sort purified naive CD4+ effectors (CD4+/CD25-/CD62Lhi) from C57BL/6 mice were cultured under TH17 polarizing conditions for 11 days. Two rounds of anti-CD3 + anti-CD28 stimulation in the presence of TH17 inducing cytokines yielded a culture in which >90% of the cells were producing IL-17 with minimal production of IFN-gamma. These cells were transferred into lethally irradiated B6D2 recipients at various doses with whole splenic T cells. The results demonstrated that a sub-lethal dose of T cells could be made lethal by substituting 1×106 naive whole T cell effectors for TH17s. Interestingly, substitution of 2×106 TH17s did not cause significant mortality, however typical signs of GVHD (hunching, weight loss, fur ruffling) were observed. In addition, transfer of TH17s alone caused significant morbidity with skin ulceration and extensive loss of fur. In a minority of mice TH17s alone were sufficient to induce lethality. To investigate the trafficking of TH17s in GVHD, we culture CD4+ naive effectors from GFP+ mice under optimal TH17 conditions and transferred these cells into irradiated B6D2 recipients. Fluorescent microscopy revealed extensive infiltration by GFP+ TH17 cells in GVHD target tissues, specifically the entire GI tract, lung, as well as liver. Additionally these cells were found extensively in secondary lymphoid organs seven days after transfer. By day 14 post-transfer the emigrated cells had undergone significant expansion. We confirmed these findings by characterizing GFP+ cells at these sites by flow cytometric analysis.CONCLUSIONS: These data demonstrate that in vitro differentiated TH17s alone can cause lethal acute GVHD and can synergize to cause lethal GVHD when given with a suboptimal dose of naive T cells. The trafficking data show that TH17s infiltrate and expand robustly in lymphoid and GVHD target organs in the first two weeks post transplanataion. Collectively these results describe a pathogenic role of TH17s in acute GVHD. [Display omitted]

Image-Guided Analyses Reveal that Non-CD4 Splenocytes Contribute to CD4+T Cell–Mediated Inflammation Leading to Islet Destruction by Altering Their Local Function and Not Systemic Trafficking Patterns

Recruitment of CD4(+) T cells into islets is a critical component of islet inflammation (insulitis) leading to type 1 diabetes; therefore, determining if conditions used to treat diabetes change their trafficking patterns is relevant to the outcome. Cotransfer of CD4(+)BDC2.5 (BDC) cells with non-CD4 splenocytes obtained from newly diabetic NOD mice, but not when they are transferred alone, induces accelerated diabetes. It is unclear whether these splenocytes affect diabetes development by altering the systemic and/or local trafficking and proliferation patterns of BDC cells in target and nontarget tissues. To address these questions, we developed an animal model to visualize BDC cell trafficking and proliferation using whole-body in vivo bioluminescence imaging and used the images to direct tissue sampling for further analyses of the cell distribution within tissues. The whole-body, or macroscopic, trafficking patterns were not dramatically altered in both groups of recipient mice. However, the local patterns of cell distribution were distinct, which led to invasive insulitis only in cotransferred mice with an increased number of islet-infiltrating CD11b(+) and CD11c(+) cells. Taken together, the non-CD4 splenocytes act locally by promoting invasive insulitis without altering the systemic trafficking patterns or proliferation of BDC cells and thus contributing to diabetes by altering the localization within the tissue.

Recruitment and proliferation of CD4+ T cells in synovium following adoptive transfer of adjuvant-induced arthritis

Adjuvant-induced arthritis can be transferred to naive Dark Agouti (DA) strain (DA.CD45.1) rats by thoracic duct (TD) lymphocytes. Disease can be re-induced in convalescent rats by further transfer of arthritogenic cells, suggesting that resolution of the adoptive disease is not due to active regulation. To examine whether resolution is due to exhaustion of effector cells, we transferred the disease to DA.CD45.1 recipients, using CD4+ T cells from DA.CD45.2 donors. At the height of the adoptively transferred disease, donor cells comprised only 5-10% of recirculating CD4+ T cells but they accounted for approximately 40% of the CD4+ T cells in synovium-rich tissues of the hind paws. Approximately 65% of the donor cells in the synovium expressed a marker of proliferation (Ki-67 antigen). Division of CD4+ T cells continued in shielded paws after suppression of the recirculating pool of lymphocytes by selective irradiation. Intravenously injected CD4+ TD T lymphoblasts from arthritic donors were recruited to normal paws and, in greater numbers, to paws of animals with existing arthritis. Survival of the [125I]iodo-deoxyuridine-labeled lymphoblasts was greater in animals with existing arthritis. We conclude that effector CD4+ T cells in target tissues can proliferate in response to autoantigens and exhibit enhanced survival. However, without a continuous supply, adoptively transferred effector cells do not produce autonomous local disease, due to limits to their lifespan and ability to replicate indefinitely.

140. Primary Adenovirus Specific Cytotoxic T Lymphocyte Response Occurs after Viral Clearance and Liver Enzyme Elevation

Top of pageAbstract The relative contribution of the specific cytotoxic T lymphocyte (CTL) response and innate immune response to the clearance of adenovirus (Ad) and its transgene in the context of gene therapy has been a subject of debate. Transient transgene expression was believed due to the elimination of Ad transduced cells by CTLs. To determine the relative contribution of specific CTL killing of Ad-infected hepatocytes, we have utilized a wild-type Ad5 to study viral clearance from the liver and its associated liver enzyme elevation during a primary immune response. Human Ad5 replicates poorly in mice due to species restriction but induces a dominant CTL response to the E1B peptide (AA192 VNIRNCCYI) associated with mouse class I MHC antigen, H2-Db, in C57BL/6 (B6) mice. Thus, Ad5 was used to mimic a low replicative Ad vectors used in humans and an MHC-I tetramer that contains the E1Bp, H2-Db and b2-microglubulin (Db-E1B), was developed and used to detect the Ad-specific CTLs in Ad5-primed mice. After Ad5 infection, the Db-E1B+CD8+T cells were undetectable in the spleen, liver and lung until day 5, and peaked at day 8. At the peak of the CTL response, 8 days post infection, 10% of CD8+ cells in the spleen. Surprisingly, 45% in the liver and 55% in the lung of the CD8+ T cells were Db-E1B+, indicating that most CD8+ T cells in target tissue are specifically recruited to these sites. Furthermore, nearly 100% of Db-E1B+CD8+ cells were IFN|[gamma]| and granzyme-B positive with intracellular staining, indicating that the recruited CD8+CTL exhibit potent killing activity. The E1B-specific in vivo CTL response was detected by the transfer of CFSE-labeled, E1B192 peptide-pulsed target cells from H-2b mice into Ad5-primed syngeneic mice. The magnitude of the in vivo CTL response correlated with the number of Db-E1B+ CTLs in the spleen and liver (r2=0.96, p<0.01). Interestingly, viral DNA genome and E1B mRNA peaked at day 2 and reduced rapidly by day 6 and then maintained at a stable level without significant change 200 days after infection. Viral clearance correlated with the peak liver enzyme response that occurred on day 4, before CTLs became detectable. In contrast, there was no further change in the level of viral DNA genome and E1B mRNA in the liver or lung as a result of the peak CTL response at day 8. Administration of Ad5 to athymic SCID mice induced no CD8 T cells or in vivo specific CTL response against E1B192 peptide, yet viral clearance and liver enzyme response in SCID mice were comparable to those of B6 mice. Taken together, our results suggest that the innate response, but not the CTL response, plays a dominant role in elimination of Ad during a primary response. Stable long-term expression of lower levels of virus and transgene persist for 200 days despite the presence of potent CD8+ CTLs, indicating that they reside in liver cells that are resistant to CTL elimination. These findings will be useful in developing efficient and safe Ad gene therapy protocols.

Association of Fetal Hormone Levels with Stem Cell Potential: Evidence for Early Life Roots of Human Cancer

Intrauterine and perinatal factors have been linked to risk of childhood leukemia, testicular cancer, and breast cancer in the offspring. The pool of stem cells in target tissue has been suggested as a critical factor linking early life exposures to cancer. We examined the relation between intrauterine hormone levels and measurements of stem cell potential in umbilical cord blood. Cord blood donors were 40 women, ages >/=18 years, who delivered, from August 2002 to June 2003, a singleton birth after a gestation of at least 37 weeks. We assayed plasma concentrations of estradiol, unconjugated estriol, testosterone, progesterone, prolactin, sex hormone binding globulin, insulin-like growth factor-I (IGF-I), and IGF binding protein-3. For stem cell potential, we measured concentrations of CD34(+) and CD34(+)CD38(-) cells and granulocyte-macrophage colony-forming unit (CFU-GM). We applied linear regression analysis and controlled for maternal and neonatal characteristics. We found strong positive associations between IGF-I and stem cell measures, 1 SD increase in IGF-I being associated with a 41% increase in CD34(+) (P = 0.008), a 109% increase in CD34(+)CD38(-) (P = 0.005), and a 94% increase in CFU-GM (P = 0.01). Similar associations were observed for IGF binding protein-3. Among steroid hormones, estriol and testosterone were significantly positively associated with CD34(+) and CFU-GM. These findings indicate that levels of growth factors and hormones are strongly associated with stem cell potential in human umbilical cord blood and point to a potential mechanism that may mediate the relationship between in utero exposure to hormones and cancer risk in the offspring.

Developmental ontogeny of prolactin and its receptor in fish

Prolactin (PRL) is a member of a family of structurally similar proteins which includes growth hormone (GH) and somatolactin (SL) in teleost fish. The genes encoding these proteins are expressed principally in the pituitary gland and sequence analysis reveals they share considerable similarity. GH, PRL, and SL bring about their physiological action by binding to specific receptors localised in the membrane of cells in target tissue. The PRL receptor (PRLR) and GH receptor (GHR) have been identified in a number of teleosts but the SL receptor remains to be characterised. On hormone binding, receptors dimerise, and signal transduction occurs via the JAK/STAT signalling pathway. The principal action of PRL in fish is freshwater osmoregulation, although it has also been implicated in reproduction, behaviour, growth, and immunoregulation. The role of PRL in early development and metamorphosis is well established, respectively, in mammals and amphibians, although its role in fish is not so well known. Studies have shown that PRL mRNA and protein are restricted to the developing pituitary gland in fish embryos and larvae. PRLR mRNA and protein is also present in fish embryos and has a widespread tissue distribution in larvae. The levels of PRLR and PRL mRNA vary throughout embryonic and early larval development. The potential role of PRL in fish embryos and larvae is considered in relation to their physiological status.

The ICOS:ICOSL Costimulatory Pathway Plays an Important Role in GVHD and Bone Marrow (BM) Graft Rejection.

ICOS, a CD28/CTLA-4 family member, is expressed on activated T cells. ICOS Ligand, a B7 family member, is constitutively expressed on B cells, monocytes and some T cells. Through the use of blocking anti-ICOS mAb and ICOS deficient (−/−) mice, we found that ICOS:ICOSL interactions play an important role in GVHD and BM graft rejection. Anti-ICOS mAb (given d-1 to d28 post BMT) significantly delayed or reduced mortality at 2 different T cell doses in a full MHC-disparate GVHD model. ICOS−/− T cells led to delayed or reduced mortality at 3 different cell doses compared to wild-type T cells. ICOS−/− CD4+ or CD8+ T cells infused into class II- or class I-disparate recipients, respectively, revealed that ICOS:ICOSL interactions regulate both CD4+ and CD8+ T cell alloresponses. Anti-ICOS inhibited GVHD in a CD28-independent fashion. Anti-ICOS inhibited GVHD mediated by either stat 4−/− or stat 6−/− T cells indicating that the ICOS pathway regulates both Th2 and Th1-mediated GVHD. In contrast to blockade of the B7:CD28/CTLA-4, CD40L:CD40 or the OX40:OX40L pathway, anti-ICOS mAb inhibited GVHD even when delayed until d5 post BMT, a time when substantial T cell expansion has occurred. A TCR transgenic model of GVHD was used to further study effects of ICOS:ICOSL blockade. All CB6 F1 recipients of anti-host alloreactive 2C CD8+ and TEa CD4+ T cells succumbed to GVHD mortality by d18 after transfer of cells. In contrast, 88% of anti-ICOS-treated mice survived long-term. Evaluation of spleens early after transplant revealed that anti-ICOS mAb reduced the number of TEa CD4+ cells by 44% and 2C CD8+ cells by 83%. Green fluorescent protein (GFP) 2C CD8+ and GFP TEa CD4+ T cells were infused into irradiated CB6 F1 mice and irrelevant or anti-ICOS mAb was administered. Mice were imaged on d4, 7 and 12 after T cell transfer. By d7, pronounced infiltration of GFP+ cells was noted in the peripheral and mesenteric LN, spleen, Peyer's patches (PP), skin, gingiva, liver, kidney, lung, ileum, and colon of GVHD control mice. In contrast, there were fewer GFP+ cells in the spleen, ileum, colon, kidney, lung, skin and gingiva of anti-ICOS-treated mice, although there was no decrease in GFP+ cells in LNs or PP. To study the role of host ICOS expression in BM graft rejection, wild-type or ICOS−/− mice were sublethally irradiated and given allogeneic BM and evaluated for donor chimerism at 6 weeks post BMT. Five of 10 wild type mice engrafted (ave − 26% donor) in contrast to all 10 of ICOS−/− mice (ave − 71% donor). Collectively, these data indicate that ICOS:ICOSL interactions play an important role in GVHD, whether mediated by CD4+ Th1 or Th2 T cells or CD8+ T cells. Importantly, blockade of ICOS:ICOSL after initiation of alloresponses inhibited GVHD, in contrast to blockade of other costimulatory pathways, suggesting that the ICOS pathway may be a novel therapeutic target in primed transplantation situations. Anti-ICOS interfered with expansion of donor T cells in the spleen early after transplant and reduced the number of effector cells in several GVHD target tissues. These data suggest this pathway may be indicated for therapeutic targeting for the inhibition of GVHD and BM graft rejection.

Update on graft versus host disease.

Update on graft versus host disease.

Metabolic, Biochemical and Histopathological Studies to Explore the Calcium Role in Antagonizing Gentamicin . Toxic Side Effect in Rats

Twenty rats were divided equally into four groups, housed separately in special metabolic cages. Animals of first, second and third group injected S.C. with gentamicin sulphate alone at a dose of 5mg/Kg (T) or as a mixture with calcium at a dose of 7.5 mg/Kg injected S.C. (T2) or separately with calcium given orally at a dose of 22.5 mg/Kg (T3). The fourth group considered as a control and injected S.C. with distilled water (C). The experiment includes samples analysis of sample of one week pre and post treatment and three weeks daily treatment. &#x0D; The results showed significant changes in studied parameters proportional with treatment period in animals of first group (Ti) in comparison with other groups expressed metabolically as a decline in body weight, increase in daily urine volume, biochemical as increase in serum creatinine, BUN, AP, AST, as well as histopathological changes in kidney nephrons and tubules. &#x0D; Calcium therapy whether as a mixture (T2) or alone (T3) gave nearly complete protection against gentamicin toxicity in the first and second week and incomplete one in the third week of treatment that could be attributed to the competitive interaction between calcium and gentamicin due to the similarity in charges and binding site that may cause a decline in gentamicin disposition in the target organ and tissue cell and so reduce its toxic side effect.

A neuroendocrine-immune interface. The immunostimulatory state of aldosteronism.

Hypertension is an important determinant of heart failure. Ventricular systolic and/or diastolic dysfunction can eventuate in an activation of the renin-angiotensin-aldosterone system (RAAS). Circulating RAAS effector hormones lead to: the appearance of the congestive heart failure syndrome; and a systemic illness that features oxi/nitrosative stress in various tissues, including blood, together with circulating pro-inflammatory cytokines, anorexia and, ultimately, cachexia. In addition to its well-known endocrine properties, expressed in epithelial cells of classic target tissues, aldosterone (ALDO) has an emerging portfolio of actions in nonclassic target tissues, such as circulating lymphocytes and monocytes (or peripheral blood mononuclear cells [PBMC]). This neuroendocrine-immune interface is based on Na(+)-dependent, ALDO-induced iterations in PBMC cytosolic free [Mg(2+)](i) and [Ca(2+)](i). Ca(2+) loading contributes to an induction of oxi/nitrosative stress and activation of PBMC transcriptome. This immunostimulatory state begets a pro-inflammatory/fibrogenic vascular phenotype involving the heart and systemic organs and can be prevented by either ALDO receptor antagonism or antioxidant. The established efficacy of ALDO receptor antagonism as an integral component of the overall management of symptomatic heart failure may include its immunomodulatory properties. This brief review traces studies that led to and then identified the neuroendocrine-immune interface that accompanies aldosteronism.

Anti-inflammatory potential of the selective phosphodiesterase 4 inhibitor N-(3,5-dichloro-pyrid-4-yl)-[1-(4-fluorobenzyl)-5-hydroxy-indole-3-yl]-glyoxylic acid amide (AWD 12-281), in human cell preparations.

AWD 12-281 is a potent (IC(50) = 9.7 nM) and highly selective inhibitor of the phosphodiesterase 4 (PDE4) isoenzyme with low affinity to the high-affinity rolipram-binding site. The compound was optimized for topical treatment of asthma, chronic obstructive pulmonary disease (COPD), and allergic rhinitis. The aim of the present study was to assess the effect of AWD 12-281 in human inflammatory cells. Peripheral blood mononuclear cells (PBMCs), diluted whole blood, and human nasal polyp cells derived from surgically resected nasal polyps from patients with polyposis comprise sources of target tissue cells that can be used to predict anti-inflammatory effects in patients. AWD 12-281 was capable of suppressing the production of cytokines in stimulated PBMCs: interleukin-2 (IL-2, phytohemagglutinin stimulation), IL-5 (concanavalin A stimulation), IL-5 and IL-4 (anti-CD3/anti-CD28 costimulation), and lipopolysaccharide-stimulated release of tumor necrosis factor alpha (TNF alpha). The corresponding values for half-maximum inhibition, EC(50), for AWD 12-281 were within a narrow range (46-121 nM). Comparing the effect of AWD 12-281 with roflumilast, cilomilast (SB 207499), rolipram (RPR-73401), and 1-(3-nitrophenyl)-3-(4-pyridylmethyl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (RS-25344-000), it could be shown that the PDE4 inhibitory activity was closely correlated with inhibitory potential as measured by the above-described assays. AWD 12-281 was also shown to suppress TNF alpha release in dispersed nasal polyps (EC(50) = 111 nM) and in diluted whole blood (EC(50) = 934 nM). The reduced activity in human blood may be related to high plasma protein binding. Currently, phase II clinical studies are under way to evaluate the therapeutic potential of AWD 12-281 in asthma, COPD, and allergic rhinitis.

Trypanosoma cruzi infection: a continuous invader-host cell cross talk with participation of extracellular matrix and adhesion and chemoattractant molecules.

Several lines of evidence have shown that Trypanosoma cruzi interacts with host extracellular matrix (ECM) components producing breakdown products that play an important role in parasite mobilization and infectivity. Parasite-released antigens also modulate ECM expression that could participate in cell-cell and/or cell-parasite interactions. Increased expression of ECM components has been described in the cardiac tissue of chronic chagasic patients and diverse target tissues including heart, thymus, central nervous system and skeletal muscle of experimentally T. cruzi-infected mice. ECM components may adsorb parasite antigens and cytokines that could contribute to the establishment and perpetuation of inflammation. Furthermore, T. cruzi-infected mammalian cells produce cytokines and chemokines that not only participate in the control of parasitism but also contribute to the establishment of chronic inflammatory lesions in several target tissues and most frequently lead to severe myocarditis. T. cruzi-driven cytokines and chemokines may also modulate VCAM-1 and ICAM-1 adhesion molecules on endothelial cells of target tissues and play a key role in cell recruitment, especially of activated VLA-4+LFA-1+CD8+ T lymphocytes, resulting in a predominance of this cell population in the inflamed heart, central nervous system and skeletal muscle. The VLA-4+-invading cells are surrounded by a fine network of fibronectin that could contribute to cell anchorage, activation and effector functions. Since persistent "danger signals" triggered by the parasite and its antigens are required for the establishment of inflammation and ECM alterations, therapeutic interventions that control parasitism and selectively modulate cell migration improve ECM abnormalities, paving the way for the development of new therapeutic strategies improving the prognosis of T. cruzi-infected individuals.

A class B scavenger receptor mediates the cellular uptake of carotenoids in Drosophila.

Carotenoids are currently being intensely investigated regarding their potential to lower the risk of chronic disease and vitamin A deficiency. Invertebrate models in which vitamin A deficiency is not lethal allow the isolation of blind but viable mutants affected in the pathway leading from dietary carotenoids to vitamin A. Using a mutant in one of these model systems, Drosophila, the vitamin A-forming enzyme has recently been molecularly identified. We now show that the molecular basis for the blindness of a different Drosophila mutant, ninaD, is a defect in the cellular uptake of carotenoids. The ninaD gene encodes a class B scavenger receptor essential for the formation of the visual chromophore. A loss of this function results in a carotenoid-free and thus vitamin A-deficient phenotype. Our investigations provide molecular insight into how carotenoids may be distributed into cells of target tissues in animals and indicate a crucial role of class B scavenger receptors rendering dietary carotenoids available for subsequent cell metabolism, as needed for their various physiological functions.

Formaldehyde cytotoxicity in three human cell types assessed in three different assays

International standards for preclinical screening of the cytotoxicity of dental materials so far recommend the use of established cell lines. The aim of this study was to assess the relative susceptibility of human dental pulp fibroblasts (HPF), human buccal epithelial cells (HBE) and HeLa cervix cancer cells exposed to identical cytotoxic challenges. Formaldehyde, which may be released from dental materials such as dental composites, glassionomer cements, and endodontic sealers, was used as test chemical. Cytotoxicity data including dose–response relations and TC 50 values were assessed in three different assays: BrdU incorporation, neutral red uptake and MTT assays. HBE and HPF demonstrated statistically significant lower TC 50 values in both the neutral red and the BrdU assay in comparison to HeLa cells. In the MTT assay no statistically significant differences were observed between the cell types. In the two target-tissue cell types (HPF and HBE) the Neutral Red assay revealed lower TC 50 values in comparison to the BrdU assay. In HeLa cells no statistically significant differences were observed between the assays. In conclusion, the present study confirms that cytotoxicity data obtained by cell culture studies are influenced by both cell culture model and choice of assay. Under identical experimental conditions, human target tissue cells appeared to be more sensitive to formaldehyde toxicity than human HeLa cancer cells.