Human basophils activated by mast cell-derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid

The vitamin A metabolite retinoic acid (RA) plays a fundamental role in cellular functions by activating nuclear receptors. Retinaldehyde dehydrogenase-II (RALDH2) creates localized RA gradients needed for proper embryonic development, but very little is known regarding its regulated expression in adults. Using a human ex vivo model of allergic inflammation by coincubating IgE receptor-activated mast cells (MCs) with blood basophils, we observed prominent induction of a protein that was identified as RALDH2 by mass spectroscopy. RALDH2 was selectively induced in basophils by MC-derived interleukin-3 (IL-3) involving PI3-kinase and NF-kappaB pathways. Importantly, neither constitutive nor inducible RALDH2 expression was detectable in any other human myeloid or lymphoid leukocyte, including dendritic cells. RA generated by RALDH2 in basophils modulates IL-3-induced gene expression in an autocrine manner, providing positive (CD25) as well as negative (granzyme B) regulation. It also acts in a paracrine fashion on T-helper cells promoting the expression of CD38 and alpha4/beta7 integrins. Furthermore, RA derived from IL-3-activated basophils provides a novel mechanism of Th2 polarization. Thus, RA must be viewed as a tightly controlled basophil-derived mediator with a high potential for regulating diverse functions of immune and resident cells in allergic diseases and other Th2-type immune responses.

Soluble TNF-alpha but not transmembrane TNF-alpha sensitizes T cells for enhanced activation-induced cell death

In addition to its proinflammatory effects, TNF-alpha exhibits immunosuppression. Here, we compared the capacities of transmembrane TNF-alpha (tmTNF) and soluble TNF-alpha (sTNF) in regulating expansion of activated T cells by apoptosis. Splenic CD4(+) T cells from wtTNF, TNF-alpha-deficient (TNF(-/-)) and TNF(-/-) mice expressing a non-cleavable mutant tmTNF showed comparable proliferation rates upon TCR-mediated stimulation. Activation-induced cell death (AICD), however, was significantly attenuated in tmTNF and TNF(-/-), compared with wtTNF CD4(+) T cells. Addition of sTNF during initial priming was sufficient to enhance susceptibility to AICD in tmTNF and TNF(-/-) CD4(+) T cells to levels seen in wtTNF CD4(+) T cells, whereas addition of sTNF only during restimulation failed to enhance AICD. sTNF-induced, enhanced susceptibility to AICD was dependent on both TNF receptors. The reduced susceptibility of tmTNF CD4(+) T cells for AICD was also evident in an in vivo model of adoptively transferred CD4(+) T-cell-mediated colonic inflammation. Hence, the presence of sTNF during T-cell priming may represent an important mechanism to sensitize activated T cells for apoptosis, thereby attenuating the extent and duration of T-cell reactivities and subsequent T-cell-mediated, excessive inflammation.

ABO blood group incompatible haematopoietic stem cell transplantation and xenograft rejection

The current organ shortage in transplantation medicine stimulates the exploration of new strategies to expand the donor pool including the utilisation of living donors, ABO-incompatible grafts, and xenotransplantation. Preformed natural antibodies (Ab) such as anti-Gal or anti-A/B Ab mediate hyperacute graft rejection and thus represent a major hurdle to the employment of such strategies. In contrast to solid organ transplantation (SOT), ABO blood group incompatibilities are of minor importance in haematopoietic stem cell transplantation (HSCT). Thus, ABO incompatible HSCT may serve as an in vivo model to study carbohydrate antigen (Ag)-mismatched transplantations such as ABO-incompatible SOT or the effect of preformed Ab against Gal in xenotransplantation. This mini-review summarises our clinical and experimental studies performed with the support of the Swiss National Science Foundation program on Implants and Transplants (NFP-46). Part 1 describes data on the clinical outcome of ABO-incompatible HSCT, in particular the incidence of several immunohaematological complications, acute graft-versus-host-disease (GvHD), and the overall survival. Part 2 summarises the measurements of anti-A/B Ab in healthy blood donors and ABO-incompatible HSCT using a novel flow cytometry based method and the potential mechanisms responsible for the loss of anti-A/B Ab observed following minor ABO-incompatible HSCT, ie the occurrence of humoral tolerance. Part 3 analyses the potential of eliminating Gal expression as well as specific complement inhibitors such as dextran sulfate and synthetic tyrosine analogues to protect porcine endothelial cells from xenoreactive Ab-mediated damage in vitro and in a hamster-to-rat heart transplantation model. In conclusion, due to similarities of the immunological hurdles of ABO incompatible transplantations and xenotransplantation, the knowledge obtained from both fields might lead to new strategies to overcome humoral rejection in transplantation.

The effect of pulsed jet lavage in vertebroplasty on injection forces of PMMA bone cement: an animal study

Percutaneous vertebroplasty, comprising of the injection of polymethylmethacrylate (PMMA) into vertebral bodies, is an efficient procedure to stabilize osteoporotic compression fractures as well as other weakening lesions. Besides fat embolism, cement leakage is considered to be one of the major and most severe complications during percutaneous vertebroplasty. The viscosity of the PMMA during injection plays a key role in this context. It was shown in vitro that the best way to lower the risk of cement leakage is to inject the cement at higher viscosity, which is requires high injection forces. Injection forces can be reduced by applying a newly developed lavage technique as it was shown in vitro using human cadaver vertebrae. The purpose of this study was to prove the in vitro results in an in vivo model. The investigation was incorporated in an animal study that was performed to evaluate the cardiovascular reaction on cement augmentation using the lavage technique. Injection forces were measured with instrumentation for 1 cc syringes, additionally acquiring plunger displacement. Averaged injection forces measured, ranged from 12 to 130 N and from 28 to 140 N for the lavage group and the control group, respectively. Normalized injection forces (by viscosity and injection speed) showed a trend to be lower for the lavage group in comparison to the control group (P = 0.073). In conclusion, the clinical relevance on the investigated lavage technique concerning lowering injection forces was only shown by trend in the performed animal study. However, it might well be that the effect is more pronounced for osteoporotic vertebral bodies.

Taurolidine in the prevention and therapy of lung metastases

OBJECTIVE: During surgery for colon carcinoma, tumour cells may spread into the blood and may lead to the development of distant metastases. The most frequent sites of metastases are the liver and lungs. A new therapeutic approach is required to prevent tumour implantation of freely circulating tumour cells during and after surgery and to treat established metastases. The aim of this prospective study was to observe the influence of long-term intravenous taurolidine on the development of lung metastases after intravenous injection of colon adenocarcinoma cells. METHODS: Tumour cells (DHD/K12/TRb colon adenocarcinoma cell line, 1 x 10(6) cells) were injected into the right vena jugularis interna of BDIX rats. The animals (n=13) were randomised into three groups: group 1: tumour cell implantation without taurolidine application (control group); group 2: tumour cell implantation and simultaneous start of the taurolidine injection through osmotic pump, removal of the osmotic pump on day 7; group 3: tumour cell implantation on day 0 and start of the taurolidine injection through osmotic pump on day 14. RESULTS: In the taurolidine groups, the number and size of lung metastases were significantly lower compared to the control group (p=0.018; p=0.018 and p=0.036; p=0.018). Although the results of the intravenous long-term therapy with taurolidine in group 2 did not reach statistical significance in comparison with the results of group 3, a positive trend was revealed: The mean number of metastases in group 2 was 18.2 versus 28.2 in group 3. CONCLUSIONS: The application of taurolidine tends to prevent the development of lung metastases. Furthermore, taurolidine seems to reduce established lung metastases in this in vivo model. Taurolidine may offer additional therapeutic options in patients with colon adenocarcinoma.

Experimentally induced cam impingement in the sheep hip

Sheep hips have a natural non-spherical femoral head similar to a cam-type deformity in human beings. By performing an intertrochanteric varus osteotomy, cam-type femoro-acetabular impingement (FAI) during flexion can be created. We tested the hypotheses that macroscopic lesions of the articular cartilage and an increased Mankin score (MS) can be reproduced by an experimentally induced cam-type FAI in this ovine in vivo model. Furthermore, we hypothesized that the MS increases with longer ambulatory periods. Sixteen sheep underwent unilateral intertrochanteric varus osteotomy of the hip with the non-operated hip as a control. Four sheep were sacrificed after 14, 22, 30, and 38-weeks postoperatively. We evaluated macroscopic chondrolabral alterations, and recorded the MS, based on histochemical staining, for each ambulatory period. A significantly higher prevalence of macroscopic chondrolabral lesions was found in the impingement zone of the operated hips. The MS was significantly higher in the acetabular/femoral cartilage of the operated hips. Furthermore, these scores increased as the length of the ambulatory period increased. Cam-type FAI can be induced in an ovine in vivo model. Localized chondrolabral degeneration of the hip, similar to that seen in humans (Tannast et al., Clin Orthop Relat Res 2008; 466: 273-280; Beck et al., J Bone Joint Surg Br 2005; 87: 1012-1018), can be reproduced. This experimental sheep model can be used to study cam-type FAI.

EFFICACY AND MECHANISM OF â-DEFENSIN2 FUSED ANTIGEN PROTEIN VACCINES

Vaccines which use the strategy of fusing adjuvant murine â-defensin2 (mBD2) to an antigen in order to elicit stronger anti-antigen immune responses are referred to as murine â-defensin2 (mBD2) vaccines. Previous studies have validated the potential of mBD2 vaccines, thus in this study we focus on increasing vaccine efficacy as well as mechanism elucidation. Initially, we demonstrate superior IFN-ã release levels by antigen specific effector T cells when antigen is crosspresented by dendritic cells (DC) which absorbed mBD2 vaccine (mBD2 fused antigen protein) over antigen alone. We move unto an in vivo model and note significant increases in the expansion of antigen specific class I T cells but not class II T cells when receiving mBD2 vaccine over antigen alone. Further, knowing mBD2’s link with CC chemokine receptor 6 (CCR6) and Toll-like receptor 4 (TLR4) we note that this enhanced class I T cell expansion is CCR6 independent but TLR4 dependent. With anti-tumor responses desired, we demonstrate in tumor protection experiments with mice, compelling tumor protection when combining adoptive T cell therapy and mBD2 vaccine immunization. We further note that mBD2 vaccines are not limited by the antigen and characterize a viable strategy for enhancing tumor antigen immunogenicity.

Effects of gossypol on DNA replication in mammalian cells

Gossypol, a binaphthalene compound, possesses male infertility effects. However, its mechanism of action and effects on somatic cells are not yet understood. The purpose of this study was to examine the effects of gossypol on mammalian cell growth and DNA replication, using tissue culture cells (HeLa) as an in vivo model.^ Gossypol inhibited DNA synthesis in HeLa cells at low doses, without affecting RNA or protein synthesis. This caused cells to accumulate in S phase without affecting cells in other phases of the cell cycle. The inhibition of DNA synthesis was both dose- and time-dependent. This irreversible block was associated with a decrease in HeLa plating efficiency. Gossypol did bind to DNA but did not measurably affect its ability to serve as a template for DNA polymerase $\alpha$, the major replicative enzyme. Only in the absence of serum could gossypol induce single-strand DNA breaks in HeLa cells; no DNA-DNA or DNA-protein crosslinks were formed.^ Gossypol exhibited dose-dependent inhibition of a number of eukaryotic and prokaryotic replicative DNA polymerases both in vitro and in vivo. This inhibition was kinetically non-competitive with respect to the DNA template and dNTP substrates. Both a filter binding assay and polyacrylamide gel electrophoresis were used to study gossypol binding to DNA polymerase. Inhibition resulted from drug binding to two adjacent amino acid residues on the enzyme. Binding was found to be irreversible and mediated through either non-covalent interactions or by Schiff's base formation between the aldehyde groups of gossypol and the $\varepsilon$-NH$\sb2$ groups of amino acid residues on the polymerase. Structure-function studies using eleven gossypol derivatives revealed that both aldehyde and hydroxyl groups function independently to effect inhibition of DNA polymerase and DNA replication. The activities of DNA polymerase $\beta$ and ribonucleotide reductase were also inhibited by increasing gossypol concentrations.^ These studies demonstrate that the gossypol-mediated inhibition of DNA replication is due in part to inhibition of key replicative enzymes, such as DNA polymerase $\alpha$. The study of DNA polymerase may serve as a model for the interaction of enzymes with gossypol, a drug which may prove useful as a chemotherapeutic agent. ^

Expression and function of psoriasin (S100A7) and koebnerisin (S100A15) in the brain

The expression and function of psoriasin in the brain have been insufficiently characterized. Here, we show the induction of psoriasin expression in the central nervous system (CNS) after bacterial and viral stimulation. We used a pneumococcal meningitis in vivo model that revealed S100A15 expression in astrocytes and meningeal cells. These results were confirmed by a cell-based in vivo assay using primary rat glial and meningeal cell cultures. We investigated psoriasin expression in glial and meningeal cells using polyinosinic-polycytidylic acid, a synthetic analog of double-stranded RNA that mimics viral infection. Furthermore, previous results showed that antimicrobial peptides have not only bactericidal but also immunomodulatory functions. To test this statement, we used recombinant psoriasin as a stimulus. Glial and meningeal cells were treated with recombinant psoriasin at concentrations from 25 to 500 ng/ml. Treated microglia and meningeal cells showed phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (ERK1/2) signal transduction pathway. We demonstrated that this activation of ERK depends on RAGE, the receptor for advanced glycation end products. Furthermore, microglia cells treated with recombinant psoriasin change their phenotype to an enlarged shape. In conclusion, our results indicate an occurrence of psoriasin in the brain. An involvement of psoriasin as an antimicrobial protein that modulates the innate immune system after bacterial or viral stimulation is possible.

Real-time ultra-high optical coherence tomography monitoring of optical tissue effects caused by selective retina therapy

Purpose: Selective retina therapy (SRT) has shown great promise compared to conventional retinal laser photocoagulation as it avoids collateral damage and selectively targets the retinal pigment epithelium (RPE). Its use, however, is challenging in terms of therapy monitoring and dosage because an immediate tissue reaction is not biomicroscopically discernibel. To overcome these limitations, real-time optical coherence tomography (OCT) might be useful to monitor retinal tissue during laser application. We have thus evaluated a proprietary OCT system for its capability of mapping optical changes introduced by SRT in retinal tissue. Methods: Freshly enucleated porcine eyes, covered in DMEM upon collection were utilized and a total of 175 scans from ex-vivo porcine eyes were analyzed. The porcine eyes were used as an ex-vivo model and results compared to two time-resolved OCT scans, recorded from a patient undergoing SRT treatment (SRT Vario, Medical Laser Center Lübeck). In addition to OCT, fluorescin angiography and fundus photography were performed on the patient and OCT scans were subsequently investigated for optical tissue changes linked to laser application. Results: Biomicroscopically invisible SRT lesions were detectable in OCT by changes in the RPE / Bruch's complex both in vivo and the porcine ex-vivo model. Laser application produced clearly visible optical effects such as hyperreflectivity and tissue distortion in the treated retina. Tissue effects were even discernible in time-resolved OCT imaging when no hyper-reflectivity persisted after treatment. Data from ex-vivo porcine eyes showed similar to identical optical changes while effects visible in OCT appeared to correlate with applied pulse energy, leading to an additional reflective layer when lesions became visible in indirect ophthalmoscopy. Conclusions: Our results support the hypothesis that real-time high-resolution OCT may be a promising modality to obtain additional information about the extent of tissue damage caused by SRT treatment. Data shows that our exvivo porcine model adequately reproduces the effects occurring in-vivo, and thus can be used to further investigate this promising imaging technique.

Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals

Nrf2 is essential for protection against acute pulmonary injury in mice

Nrf2 is a member of the “cap ‘n’ collar” family of transcription factors. These transcription factors bind to the NF-E2 binding sites (GCTGAGTCA) that are essential for the regulation of erythroid-specific genes. Nrf2 is expressed in a wide range of tissues, many of which are sites of expression for phase 2 detoxification genes. Nrf2−/− mice are viable and have a normal phenotype under normal laboratory conditions. The NF-E2 binding site is a subset of the antioxidant response elements that have the sequence GCNNNGTCA. The antioxidant response elements are regulatory sequences found on promoters of several phase 2 detoxification genes that are inducible by xenobiotics and antioxidants. We report here that Nrf2−/− mice are extremely susceptible to the administration of the antioxidant butylated hydroxytoluene. With doses of butylated hydroxytoluene that are tolerated by wild-type mice, the Nrf2−/− mice succumb from acute respiratory distress syndrome. Gene expression studies show that the expression of several detoxification enzymes is altered in the Nrf2−/− mice. The Nrf2−/− mice may prove to be a good in vivo model for toxicological studies. As oxidative damage causes DNA breakage, these mice may also be useful for testing carcinogenic agents.

Intracerebral tumor-associated hemorrhage caused by overexpression of the vascular endothelial growth factor isoforms VEGF121 and VEGF165 but not VEGF189

The vascular endothelial growth factor (VEGF) has been shown to be a significant mediator of angiogenesis during a variety of normal and pathological processes, including tumor development. Human U87MG glioblastoma cells express the three VEGF isoforms: VEGF121, VEGF165, and VEGF189. Here, we have investigated whether these three isoforms have distinct roles in glioblastoma angiogenesis. Clones that overexpressed each isoform were derived and inoculated into mouse brains. Mice that received VEGF121- and VEGF165-overexpressing cells developed intracerebral hemorrhages after 60–90 hr. In contrast, mice implanted with VEGF189-overexpressing cells had only slightly larger tumors than those caused by parental cells and little evidence of hemorrhage at these early times after implantation, whereas, after longer periods of growth, enhanced angiogenicity and tumorigenicity were apparent. There was rapid blood vessel growth and breakdown around the tumors caused by cells overexpressing VEGF121 and VEGF165, whereas there was similar vascularization but no eruption in the vicinity of those tumors caused by cells overexpressing VEGF189, and none on the border of the tumors caused by the parental cells. Thus, by introducing VEGF-overexpressing glioblastoma cells into the brain, we have established a reproducible and predictable in vivo model of tumor-associated intracerebral hemorrhage caused by the enhanced expression of single molecular species. Such a model should be useful for uncovering the role of VEGF isoforms in the mechanisms of angiogenesis and for investigating intracerebral hemorrhage due to ischemic stroke or congenital malformations.

Independent regulation of JNK/p38 mitogen-activated protein kinases by metabolic oxidative stress in the liver

The stress-activated protein kinases JNK and p38 mediate increased gene expression and are activated by environmental stresses and proinflammatory cytokines. Using an in vivo model in which oxidative stress is generated in the liver by intracellular metabolism, rapid protein–DNA complex formation on stress-activated AP-1 target genes was observed. Analysis of the induced binding complexes indicates that c-fos, c-jun, and ATF-2 were present, but also two additional jun family members, JunB and JunD. Activation of JNK precedes increased AP-1 DNA binding. Furthermore, JunB was shown to be a substrate for JNK, and phosphorylation requires the N-terminal activation domain. Unexpectedly, p38 activity was found to be constitutively active in the liver and was down-regulated through selective dephosphorylation following oxidative stress. One potential mechanism for p38 dephosphorylation is the rapid stress-induced activation of the phosphatase MKP-1, which has high affinity for phosphorylated p38 as a substrate. These data demonstrate that there are mechanisms for independent regulation of the JNK and p38 mitogen-activated protein kinase signal transduction pathways after metabolic oxidative stress in the liver.

Mice transgenic for human CD4 and CCR5 are susceptible to HIV infection

HIV entry into human cells is mediated by CD4 acting in concert with one of several members of the chemokine receptor superfamily. The resistance to HIV infection observed in individuals with defective CCR5 alleles indicated that this particular chemokine receptor plays a crucial role in the initiation of in vivo HIV infection. Expression of human CD4 transgene does not render mice susceptible to HIV infection because of structural differences between human and mouse CCR5. To ascertain whether expression of human CD4 and CCR5 is sufficient to make murine T lymphocytes susceptible to HIV infection, the lck promoter was used to direct the T cell-specific expression of human CD4 and CCR5 in transgenic mice. Peripheral blood mononuclear cells and splenocytes isolated from these mice expressed human CD4 and CCR5 and were infectible with selected M-tropic HIV isolates. After in vivo inoculation, HIV-infected cells were detected by DNA PCR in the spleen and lymph nodes of these transgenic mice, but HIV could not be cultured from these cells. This indicated that although transgenic expression of human CD4 and CCR5 permitted entry of HIV into the mouse cells, significant HIV infection was prevented by other blocks to HIV replication present in mouse cells. In addition to providing in vivo verification for the important role of CCR5 in T lymphocyte HIV infection, these transgenic mice represent a new in vivo model for understanding HIV pathogenesis by delineating species-specific cellular factors required for productive in vivo HIV infection. These mice should also prove useful for the assessment of potential therapeutic and preventative modalities, particularly vaccines.