Pulmonary function changes in experimental graft-versus-host disease of the lung.

Pulmonary graft-versus-host disease (p-GVHD) is a serious complication after allogeneic stem cell transplantation (allo-SCT) of high morbidity and high mortality. We characterized breathing patterns and pulmonary function changes in correlation to lung histopathology and survival by using a well-established murine model of p-GVHD. Lethally irradiated B6D2F1 mice received SCT from either syngeneic B6D2F1 or allogeneic C57BL/6 animals. Within 6 weeks, severe p-GVHD developed in allogeneic recipients characterized by progressive interstitial, alveolar, peribronchial, and periluminal inflammatory cell infiltration, whereas in syngeneic recipients lung histology remained normal. Allogeneic recipients demonstrated decreased minute ventilation (MV), reduced peak inspiratory and expiratory flow rates as early as 1 week after SCT. In addition, allo-SCT resulted in restrictive pulmonary function changes as early as 7 days after transplantation and in progressive airflow obstruction within 6 weeks. Decreased breathing abilities and pulmonary function changes of allogeneic recipients were associated with increased mortality and the severity of acute graft-versus-host disease (aGVHD). These findings show that p-GVHD can be characterized by changes in pulmonary function and functional respiratory insufficiency. Furthermore, our data strengthen the understanding, that the lung is a critical target organ of aGVHD.

Interleukin-10 does not affect IL-1-induced interleukin-6 and metalloproteinase production in human chondrosarcoma cells, SW1353.

Cartilage repair by transplantation of autologous chondrocytes is an option when restoring functional joints. Control of chondrocyte function is thus required. Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine affecting the expression of a wide range of immune mediators in hematopoietic and non-hematopoietic cells. Previous studies indicated that IL-10 has therapeutic potential in the treatment of chronic inflammatory joint disorders such as rheumatoid arthritis and osteoarthritis. IL-10 has been found to be chondroprotective by down-regulating metalloproteinase expression and by inhibiting the synthesis of pro-inflammatory cytokines, such as IL-6, in immune cells. In contrast, the effects of IL-10 on chondrocytes are poorly understood and have to be identified with regard to their future clinical use. In this study, we investigated the effects of IL-10 on the expression of cartilage-degrading mediators in the human chondrosarcoma cell line, SW1353, after exposure to IL-1, a key mediator in cartilage and bone destruction. We found a strong induction of the pro-inflammatory cytokine, IL-6, in IL-1-exposed SW1353 cells. Surprisingly, IL-10 had no effect on IL-1-induced IL-6, pro-MMP1, and pro-MMP13 secretion. Although RT-PCR analyses demonstrated the expression of both receptor chains of the IL-10 receptor complex (IL-10R1 and IL-10R2), exposure of SW1353 to IL-10 did not lead to phosphorylation of STAT3, the major transcription factor induced by IL-10. This was not due to a defect in STAT3, because stimulation with IL-6 resulted in its phosphorylation. Failure of SW1353 cells to respond to IL-10 was consistent with a deficient surface expression of IL-10R1. From these results we conclude that IL-10 does not exert its chondroprotective character on chondrocytes directly. Furthermore, the unresponsiveness of chondrocytes towards IL-10 might explain the vulnerability of joint cartilage to inflammation.

p38MAPK mediates IL-1-induced down-regulation of aggrecan gene expression in human chondrocytes.

The pleiotropic cytokine interleukin 1 (IL-1) is considered to be the principal inducer of mediators of cartilage degradation in both, osteoarthritis (OA) and rheumatoid arthritis (RA). IL-1 activates numerous signaling pathways involved in cartilage destruction and dedifferentiation of chondrocytes. In this study, we analyzed expression and functional effects of IL-1 in human chondrocytes. We found an IL-1-induced reduction in the expression of the cartilage specific proteoglycan aggrecan as an indicator for the IL-1-mediated dedifferentiation of chondrocytes. To block the IL-1-induced signaling pathways specifically, we incubated human chondrocytes and cartilage explants with IL-1 in the presence of different signal transduction inhibitors and analyzed their effect on aggrecan mRNA expression and IL-6 secretion. IL-6 has been found to act synergistically in the IL-1-induced suppression of the proteoglycan synthesis in chondrocytes. Our results led to the identification of p38MAPK and/or PI3K/JNK as being crucial for IL-1-induced IL-6 secretion by chondrocytes. IL-1-induced down-regulation of aggrecan expression was found to be mediated by p38MAPK and/or ERK1/2. The identification and characterization of these signaling pathways will enable us to develop new modulation strategies for therapeutic use in inflammatory joint diseases.

Failure of interferon-gamma and tumor necrosis factor in mediating anemia of chronic disease in a mouse model of protracted septic peritonitis.

Interferon-gamma (IFN-gamma) is considered one of the main inflammatory cytokines contributing to the generation of anemia of chronic disease (ACD). In this study, we used a previously described murine model for ACD based on sublethal cecal ligation and puncture (CLP) with ensuing protracted peritonitis. Within 2 weeks after CLP, a moderate normochromic anemia with low serum iron concentration and preserved iron stores develops, which is consistent with ACD. In order to determine whether IFN-gamma contributes to the development of ACD in vivo, we neutralized IFN-gamma after CLP shortly before and during the phase of most severe bone marrow depression in order to prevent anemia. Additionally, we studied IFN-gamma receptor-deficient mice that underwent CLP. Two weeks after CLP, we determined the red blood cell count, hemoglobin concentration, hematocrit, serum iron concentration, and iron stores in spleens of wild-type mice, IFN-gamma receptor-deficient mice, and mice after neutralization of IFN-gamma. Neutralization of IFN-gamma after CLP could not prevent mice from becoming anemic. Accordingly, IFN-gamma receptor-deficient mice developed anemia to the same extent as wild-type mice. Serum iron concentration was lowered both in IFN-gamma receptor-deficient and wild-type mice. Iron stores in untreated IFN-gamma receptor-deficient mice were elevated compared to untreated wild-type mice. After CLP both IFN-gamma receptor-deficient and wild-type mice had equally overloaded iron stores. Additional neutralization of TNF in IFN-gamma receptor-deficient mice also did not attenuate CLP-induced anemia. Our results clearly demonstrate that neither IFN-gamma alone nor in combination with TNF is a mediator of ACD in our model with transient anemia induced by protracted septic peritonitis.

Adult type granulosa cell tumor of the testis with a heterologous sarcomatous component: case report and review of the literature.

Adult testicular granulosa cell tumors are rare sex cord- stromal tumors of which only 45 have been previously reported. As compared with their ovarian counterparts, these tumors may follow a more aggressive course because the proportion of malignant cases is higher. We report here a unique case of a 78-year Caucasian with a left sided adult type granulosa cell tumor with a heterologous sarcomatous tumor component. A heterologous sarcomatous component has occasionally been observed in ovarian tumors but never in testicular granulosa cell tumors. The sarcomatous component showed a higher number of mitotic figures (1/Hpf) and a marked proliferation rate (up to 50% Ki 67 positive cells) compared with the granulosa type tumor component. CD 99 and the progesterone receptor were positive in both tumor components, inhibin and calretinin only in the granulosa cells, and pancytokeratin only in the sarcomatouse one. Key words: testis - ovary - granulosa cells - sarcoma - inhibin Runing title: testicular sarcomatous granulosa tumor. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/6959043481207016.

Hypoferraemia during the early inflammatory response is dependent on tumour necrosis factor activity in a murine model of protracted peritonitis.

On the grounds of clinical, in vitro and in vivo studies, tumour necrosis factor (TNF) is considered to be one of the inflammatory cytokines that contributes to to the generation of hypoferraemia and anaemia of inflammation (AI). We used a recently described murine model for AI and hypoferraemia, based on sublethal caecal ligation and puncture (CLP) with ensuing protracted peritonitis, to investigate the contribution of TNF to the generation of hypoferraemia. During the early inflammatory response to CLP, a marked decrease in serum iron concentration occurs within 8 h. To determine whether TNF contributes to the generation of hypoferraemia at this time point, we studied TNF-deficient mice and wild-type mice that underwent CLP. The serum iron concentration was decreased in wild-type mice whereas TNF-deficient mice maintained normal serum iron levels following CLP. Hypoferraemia in wild-type mice was accompanied by the downregulation of ferroportin 1 (Fp1) in macrophages. In the macrophages of TNF-deficient mice, Fp1 was not downregulated following CLP. The initial expression of hepcidin was detectable at the mRNA level but not at the protein level by immunohisto-chemistry in wild-type and TNF-deficient mice. Therefore, hepcidin does not appear to be involved in the regulation of early hypoferraemia. TNF appears to regulate the expression of Fp1 by transcriptional control. Our results demonstrate that TNF mediates hypoferraemia during the early inflammatory response by regulating the expression of Fp1 in macrophages.

Preventive usage of broad spectrum chemokine inhibitor NR58-3.14.3 reduces the severity of pulmonary and hepatic graft-versus-host disease.

Pulmonary graft-versus-host disease (pGVHD) is a major complication after allogeneic bone marrow transplantation (BMT), which involves donor leukocyte migration into the lung along chemokine gradients, leading to pulmonary dysfunction and respiratory insufficiency. As broad spectrum chemokine inhibitor (BSCI) NR58-3.14.3 suppresses leukocyte migration in response to various chemokines, including CCL2, CCL3, CCL5, we investigated the effects of NR58-3.14.3 on the evolution of pGVHD. Lethally irradiated B6D2F1 mice received BMT from syngeneic (B6D2F1) or allogeneic (C57BL/6) donors, and animals were treated with either NR58-3.14.3 or vehicle control from day -1 to day +14. At week 6, in allogeneic recipients that received BSCI, inflammatory cell infiltrates in the lung were decreased, and reduced histopathologic changes translated into improved pulmonary function when compared to allo-controls. Acute GVHD of the liver was also diminished, whereas no differences were seen in the gut. Alloantigen-dependent splenic T cell expansion and systemic TNF-alpha and IFN-gamma levels were comparable in NR58-3.14.3-treated animals and allo-controls. No suppressive effect of NR58-3.14.3 on CTL cytotoxicity was found, and diminished cellular infiltrates in lung and liver were most likely due to decreased migration of mononuclear cells. Therefore, novel approaches involving BSCIs may provide a promising tool in the management of pGVHD.

Preponderance of sensory versus sympathetic nerve fibers and increased cellularity in the infrapatellar fat pad in anterior knee pain patients after primary arthroplasty.

Sensory nerve fibers transmit pain perception and secrete pro-inflammatory substance P (SP). Sympathetic nerve fibers secrete anti-inflammatory norepinephrine and endogenous opioids, which inhibit pain perception in a bidirectional crosstalk with sensory fibers. In patients with anterior knee pain after primary arthroplasty of the knee (AKP), this study investigated in parallel the innervation of the infrapatellar fat pad by sensory and sympathetic nerve fibers. A total of 32 patients with osteoarthritis (OA) of the knee (n = 10), AKP after primary knee joint replacement (n = 7), and OA of the hip (n = 15) were included. Sensory nerve fibers were semiquantitatively detected by immunohistochemistry against SP, and sympathetic nerve fibers were stained with an antibody against tyrosine hydroxylase. Cellular density of the tissue was investigated by counting cell nuclei. The density of sympathetic nerve fibers in the fat tissue was similar in knee OA as compared to AKP. In the fat tissue, density of sensory substance P-positive nerve fibers was higher in AKP than in knee OA, which was not observed in the fibrosis capsule of the fat pad. The preponderance of sensory over sympathetic nerve fibers was accompanied by an increased cellular density in fat tissue in patients with AKP compared to knee OA. A positive correlation existed between cellularity and sensory nerve fiber density in fat tissue. This study revealed a preponderance of sensory over sympathetic innervation in the infrapatellar fat pad in AKP after primary arthroplasty of the knee, which possibly leads to aggravation and continuation of AKP and local inflammation.

In vivo development and long-term survival of engineered adipose tissue depend on in vitro precultivation strategy.

One strategy of adipose tissue engineering is to transplant adipocytes or adipocyte precursor cells in combination with polymeric materials. However, a satisfying formation of fat tissue and its long-term survival still remain major problems. There is increasing evidence that treatment of the cells prior to implantation plays a critical role in the success of adipose tissue growth. In a previous study, we established a model system based on 3T3-L1 cells that allows for reproducible engineering of mature, coherent adipose tissues in vitro. We utilized this model system in the current study and systematically investigated the long-term in vivo development of cellular constructs with varying stages of adipogenic development at the time point of implantation. Blank polyglycolic acid fiber meshes, scaffolds seeded with uninduced 3T3-L1 preadipocytes, and cell-polymer constructs precultivated under adipogenic conditions for 2, 9, or 35 days were implanted subcutaneously into nude mice. Histological analysis revealed that no fat formation occurred in constructs without adipogenic precultivation. Implantation of mature fat pads (35 days) resulted in adiponecrosis within the constructs. In contrast, implants with an immature phenotype at the time point of implantation (2 and 9 days) gave rise to vascularized, mature adipose tissue in vivo. Further, these engineered adipose tissues showed long-term survival in vivo over the whole investigation period of 24 weeks. The results of this study can contribute to the development of future clinical approaches as they give clear evidence which precultivation strategy promotes successful development and long-term survival of engineered adipose tissue.

Low viscosity histidine-tryptophan-ketoglutarate graft flush improves subsequent extended cold storage in University of Wisconsin solution in an extracorporeal rat liver perfusion and rat liver transplantation model.

Adequate flushing for liver donation requires large fluid volumes delivered at a high flow. This can be achieved more effectively with crystalloid solutions than with colloid-based solutions. This study examined the combination of initial histidine-tryptophan-ketoglutarate solution (HTK) graft flush and subsequent storage in University of Wisconsin solution (UW) to that of the single use of each solution. Livers from inbred Wistar rats were procured using aortic perfusion with UW or HTK for initial perfusion and reflushed after 30 minutes using either solution. In a third group, after perfusion with HTK, organs were reflushed with UW. A 60-minute in-vitro recirculating perfusion was performed after 24 hours of cold storage in the subsequent solution, as well as allotransplantation after 18 and 24 hours of cold storage. In extracorporeal perfusion, the HTK flush followed by UW storage was superior compared to the single use of either UW or HTK solution, as measured by portal venous pressure, bile flow, liver enzymes released into the effluent perfusate, glycerol leakage, and histological examinations. These data were consistent with the transplantation study. Histological damage and enzyme release after 5-day survival were lowest in the HTK flush and subsequent UW storage groups following 18 hours of cold storage; likewise, the 5-day survival was superior following 24 hours of cold storage. In conclusion, the combined use of HTK solution for initial graft rinse and subsequent storage in UW solution resulted in a cumulative protection. Choosing low-viscosity HTK solution for the initial organ flush may represent a feasible improvement in liver preservation, which also further reduces the required amount of UW solution.