A case of invasive pulmonary aspergillosis in renal failure

Sir, A 55-year-old ex-community care assistant was admitted witha 2 day history of increasing shortness of breath and productivecough. She suffered with chronic kidney impairment secondaryto reflux nephropathy, and had undergone a nephrectomy 1 yearpreviously. After gradually worsening uraemic symptoms, a peritonealdialysis catheter had been inserted uneventfully     

Relationship of interferon-gamma and neopterin levels during stimulation with alloantigens in vivo and in vitro

We have recently shown that interferon-gamma is capable of activating the key enzyme of pterin biosynthesis in macrophages. This leads to excretion of the stable degradation product neopterin. In this article we present experimental evidence suggesting that stimulation of T cells by alloantigens is associated with release of interferon-gamma--which, in the case of rejection, is locally restricted and not always detectable in the bloodstream. Neopterin induced by this lymphokine, however, readily penetrates tissue barriers and is detectable in the serum. This conclusion is based on two different sets of observations: (1) If supernatants of MLCs are compared with sera from patients with documented acute rejection episodes for their interferon-gamma and neopterin levels, a marked gradient is observed to exist between interferon levels measured in vitro and in vivo; this is not the case for neopterin for which comparable levels were seen. (2) Detection of interferon-gamma in sera of allograft recipients invariably precedes an increase of neopterin; on the other hand, increasing neopterin counts are also seen in the absence of detectable interferon-gamma levels in the serum. It thus appears that although interferon-gamma release during allograft rejection is primarily restricted to the tissue, evaluation of certain metabolites of interferon-dependent metabolic pathways enables definition of its endogenous release. Whereas interferon gamma represents a less reliable marker in the monitoring of rejection episodes, it might offer an additional means to differentiate rejection from systemic infections. Such a discrimination can not be achieved with the neopterin marker.     

Effects of gamma-linolenic acid on murine cells in vitro and in vivo

The effects of gamma-linolenic acid (GLA) on growth of cells of the continuous murine sarcoma line M52B were investigated in vitro. Prostaglandin (PG) production by these cells after GLA treatment was also measured. GLA inhibited the growth of M52B cells and became overtly toxic at high doses or after long periods of exposure to lower doses. The inhibitory effects of GLA were accompanied by an increase in PGE production by M52B cells. However, the rise in PGE was not statistically significant. Accordingly the extent to which PGE may contribute to the inhibition observed with GLA remains unclear. In order to establish whether these in vitro effects could be reproduced in vivo, athymic nude mice bearing murine sarcoma allografts were fed either standard laboratory diets or diets supplemented with 35% evening primrose oil, which contains 10% GLA. As there was no significant difference in tumour volumes between the two groups at the end of the treatment period, the oil-enriched diet was concluded to be without effect on tumour growth in this in vivo model.     

Novel in vivo murine model to study islet potency: engraftment and function

Standard islet potency testing uses transplantation of islets under the kidney capsule in diabetic severe combined immunodeficient (d-SCID) mice. Even though it is possible to achieve normoglycemia in the majority of recipients by this method, the surgical procedure, by itself, is technically difficult and associated with an appreciable mortality of animals. In addition, the spatially limited renal subcapsular site restricts the mass of islet tissue that can be transplanted. Matrigel basement membrane matrix (MATRIGEL), extracted from a mouse sarcoma, is rich in angiogenic growth factors and has been shown to support the growth of mammalian cells using murine models. In this report we demonstrate that subcutaneous islet transplantation with MATRIGEL can effectively achieve normoglycemia and that this is a simple and reproducible model for in vivo islet potency testing in d-SCID mice that overcomes many drawbacks of the conventional method of kidney subcapsular islet transplantation.     

Comparison of invasive and less-invasive techniques of cardiac output measurement under different haemodynamic conditions in a pig model

BACKGROUND AND OBJECTIVE: Despite the introduction of various less-invasive concepts of cardiac output measurement, pulmonary arterial thermodilution is still the most common measurement technique. METHODS: This prospective controlled study was designed to compare different methods of cardiac output measurement simultaneously. Pulmonary arterial thermodilution, transpulmonary thermodilution (PiCCO), trans-oesophageal echo-Doppler probe (HemoSonic) and partial carbon dioxide rebreathing technique (NICO monitor) were evaluated against a peri-aortic transit-time flow-probe as reference method in a clinically relevant animal model. After approval from the Local Ethics Committee on Animal Research, the investigations were conducted in nine anesthetized domestic pigs. Systemic haemodynamics were modulated systematically by the application of catecholamines, caval occlusion and exsanguination. Statistical analysis was performed with Bland-Altman and linear regression. RESULTS: A total of 366 paired cardiac output measurements were carried out at a reference cardiac output between 0.5 and 7 L min(-1). The correlation coefficients for pulmonary arterial and transpulmonary thermodilution against reference were 0.93 and 0.95, for trans-oesophageal Doppler and partial rebreathing technique 0.84 and 0.77. Pulmonary arterial thermodilution and transpulmonary thermodilution showed comparable bias and limits of agreement. Where HemoSonic showed an overestimation of cardiac output at a higher precision, NICO overestimated low and underestimated higher cardiac output values. CONCLUSIONS: Our data suggest that pulmonary arterial thermodilution and PiCCO may be interchangeably used for cardiac output measurement even under acute haemodynamic changes. The method described by Bland and Altman demonstrated an overestimation of cardiac output for both thermodilution methods. HemoSonic and NICO offer non-invasive alternatives and complementary monitoring tools in numerous clinical situations. Trend monitoring and haemodynamic optimizing can be applied sufficiently, when absolute measures are judged critically in a clinical context. The use of the NICO system seems to be limited during acute circulatory changes.     

Screw loosening in an in vitro mid fibular osteotomy model under dynamic loading conditions

The purpose of this study was to evaluate the effect of cyclic loading on screw fixation in an experimental bone plating model. The test specimens consist of plated porcine fibulae subjected to cyclic compressive, bending and torsional loading. Breakaway torque measurements of orthopedic screws are found to be significantly less than the screws tightening torque. The breakaway torque for a given screw and tapped bone hole is found to be consistent after repeated tightening, and is proposed as a viable approach to quantify bone screw loosening. After cyclic loading at moderate levels no screw loosening was identified, but instead an apparent paradoxical tightening, as observed in breakaway torque measurements of the screws. Cyclic loading of a plated fibular fracture was not found to cause screw loosening unless accompanied by gross failure as found under excessive load levels.     

In vitro and in vivo effects of photodynamic therapy on murine malignant melanoma

Background: The role of photodynamic therapy (PDT) in the treatment of malignant melanoma is not well defined, nor is it known whether the dark melanoma cells absorb the light used in PDT. Methods: In vitro studies: 2×10⁵ B16 murine melanoma cells were incubated with aluminum phthalocyanine (AlpcS₄, 2.5 mg/kg) and were then subjected to photoradiation (50, 100 or 200 J/cm²). Viability was then assessed.In vivo studies: Histology: C57/B1 mice received 2×10⁵ B16 cells subcutaneously and were randomized into study (PDT) and three control groups. AlpcS₄ 2.5 mg/kg was injected intraperitoneally and the mice were exposed to light (100 J/cm²). After 24 hours they were sacrificed and underwent autopsies. Survival: 40 mice were randomized into PDT (40 J/cm²) and control groups and were monitored for 50 days. Tumor growth: 40 mice were randomized into one control and three treatment groups (PDT on day 3, 6, or 12 after injection with B16 cells), and were monitored for 50 days. Temperature: Tumor temperatures before and at the end of PDT were recorded. Results: In vitro studies: PDT caused a decrease in cell viability to 15.5±0.7%, 11.5±2.1%, and 1.5±0.7% (at 50, 100, and 200 J/cm², respectively;P<.001). A significant reduction in thymidine incorporation was noted at all energy levels.In vivo studies: Histology: PDT caused massive tumor necrosis. Survival: PDT prolonged the survival of mice (41±13.4 days) compared to controls (15.8±3.8 days,P<.001). Tumor growth: 31 days after injection with B16 cells, the tumor size was 2.6±0.3 cm in the control group and 1.6±0.2, 0.9±0.3, and 1.0±0.4 cm in the PDT groups (days 3, 6 and 12, respectively;P<.01). Temperature: PDT increased skin temperature to 42.8°C±1.3°C, 45.3°C±3.5°C, and 51.7°C±2.7°C at 40, 60, and 100 J/cm², respectively (P<.01). Conclusions: Photodynamic therapy was found to have significant effects in experimental melanoma in mice. The role of PDT in human melanoma remains to be studied.Presented at the 50th Annual Cancer Symposium of The Society of Surgical Oncology, Chicago, Illinois, March 20–23, 1997.     

A simplified method for studying hypoxia and reoxygenation injury under in vitro conditions

A hypoxia chamber was constructed which allowed for the sequential sampling and blood gas analysis of buffer bathing cells in culture which were subjected to graded periods of hypoxia. Following hypoxia, the human fetal small intestinal cells (CCL-241) were placed into a normoxic environment for the remainder of a 24 h study period. A cytotoxicity assay revealed significant mortality in cells subjected to hypoxia and reoxygenation, but not in those subjected to hypoxia alone. Analysis of lactate dehydrogenase release into buffer samples also indicated a greater cellular injury among cells exposed to hypoxia and reoxygenation. Additionally, levels of lipid peroxidation products were found to be significantly elevated in cells exposed to periods of hypoxia followed by reoxygenation, but not hypoxia alone, as measured by a thiobarbituric acid fluorometric assay. This suggests that lipid peroxidation mediated by oxygen-derived free radical species is the mechanism of injury in these cells. This study demonstrates that such a chamber provides a more precise way to monitor hypoxia and is a useful tool for studying hypoxia and reoxygenation under in vitro conditions.     

Recombinant murine interferon-gamma inhibits the fusion of mouse alveolar macrophages in vitro but stimulates the formation of osteoclastlike cells on implanted syngeneic bone particles in mice in vivo

Osteoclasts are multinucleated cells that originate from the fusion of mononuclear precursors and are responsible for bone resorption. Indirect evidence from in vitro studies suggests that IFN-gamma and TNF-alpha inhibit and stimulate bone resorption, respectively, but contradictory results have emerged from the literature regarding the effects of IFN-gamma on macrophage multinucleation. Using highly sensitive model systems, the present work demonstrates that, in mice, rMuIFN-gamma inhibits the fusion of alveolar macrophages in vitro but augments the number of osteoclastlike cells on implanted syngeneic bone particles in vivo. Although rMuTNF-alpha fails to stimulate macrophage multinucleation in either system, treatment of implanted animals with rMuIFN-gamma appears to limit the inflammatory reaction and favor tissue repair.     

Prosthetic metals impair murine immune response and cytokine release in vivo and in vitro

This study was designed to investigate whether prosthetic metals adversely affect immune responses and the release of immunoregulatory cytokines in vivo and in vitro. Titanium and cobalt-chromium alloy were injected into the peritoneal cavity of female mice. At 5, 8, and 12 weeks after the injection, the levels of cobalt and chromium in the blood were significantly increased compared with the levels in control mice; the level of titanium was not significantly changed until 12 weeks. The release of interleukin-2 was significantly inhibited by cobalt-chromium particles after 3 weeks; titanium particles did not have the same effect until 8 and 12 weeks. The release of interleukin-γ was significantly inhibited by cobalt-chromium particles after 3 weeks but was not significantly inhibited by titanium particles until 12 weeks. The release of interferon-y was significantly inhibited by cobalt-chromium particles only at 12 weeks and was not inhibited by titanium particles. The proliferation of T cells was significantly inhibited by cobalt-chromium particles at 3 weeks and by titanium particles at 8 and 12 weeks, and the proliferation of B cells was significantly inhibited by cobalt-chromium particles after 3 weeks but was not inhibited by titanium particles. The production of immunoglobulin by lipopolysaccharide-stimulated B cells was also significantly reduced by cobalt-chromium particles after 3 weeks and by titanium particles at 8 and 12 weeks. The cytokine release by lymphocytes, proliferation of T and B cells, and immunoglobulin production by B cells were also significantly inhibited by titanium and cobalt-chromium particles, as well as by titanium, cobalt, and chromium ions in vitro, whereas these metals are not cytotoxic to murine lymphocytes in vitro. The data indicate that the metal-induced immunosuppression may be another important factor in the development of implant-associated infection in patients with a prosthesis.     

In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow

Efforts toward routine islet cell transplantation as a means for reversing type 1 diabetes have been hampered by islet availability as well as allograft rejection. In vitro transdifferentiation of mouse bone marrow (BM)-derived stem (mBMDS) cells into insulin-producing cells could provide an abundant source of autologous cells for this procedure. For this study, we isolated and characterized single cell-derived stem cell lines obtained from mouse BM. In vitro differentiation of these mBMDS cells resulted in populations meeting a number of criteria set forth to define functional insulin-producing cells. Specifically, the mBMDS cells expressed multiple genes related to pancreatic beta-cell development and function (insulin I and II, Glut2, glucose kinase, islet amyloid polypeptide, nestin, pancreatic duodenal homeobox-1 [PDX-1], and Pax6). Insulin and C-peptide production was identified by immunocytochemistry and confirmed by electron microscopy. In vitro studies involving glucose stimulation identified glucose-stimulated insulin release. Finally, these mBMDS cells transplanted into streptozotocin-induced diabetic mice imparted reversal of hyperglycemia and improved metabolic profiles in response to intraperitoneal glucose tolerance testing. These results indicate that mouse BM harbors cells capable of in vitro transdifferentiating into functional insulin-producing cells and support efforts to derive such cells in humans as a means to alleviate limitations surrounding islet cell transplantation.     

A novel bioluminescent tumor model of human renal cancer cell lines: an in vitro and in vivo characterization

PURPOSE: Bioluminescent imaging permits sensitive in vivo detection and quantification of cells engineered to emit light. We developed a bioluminescent human renal cancer cell line for in vitro and in vivo studies. MATERIAL AND METHODS: The 2 human renal cell carcinoma cell lines SN12-C and SN12-L1 were stably transfected to constitutively express luciferase using a retroviral shuttle. The bioluminescent signal was correlated with tumor cell numbers in vitro. Parental and transfected cells were compared by growth kinetics and histology. Tumor burden after heterotopic injection in immune deficient mice was monitored up to 39 days. The kinetics of the bioluminescent signal was evaluated for 1 to 60 minutes following luciferin injection. RESULTS: Bioengineered renal cancer cell lines stably expressed luciferase. The growth kinetics of the cells in vitro and the histology of tumors resulting from implantation of these cells were unaffected by retroviral transfection with the luciferase gene. As few as 1,000 cells could be reliably detected. The intensity of the bioluminescent signal correlated with the number of tumor cells in vitro. Photon emission in vivo and ex vivo correlated significantly with tumor weight at sacrifice. After intraperitoneal injection of luciferin there was a time dependent change in the intensity of the bioluminescent signal with maximum photon emission at 20 minutes (optimal 17 to 25). CONCLUSIONS: Luciferase transfected human renal cancer lines allow reliable, rapid, noninvasive and longitudinal monitoring of tumor growth in vivo. The ability to assess tumor development in vivo with time is economical and effective compared to end point data experiments.     

In vitro and in vivo activity of two second generation platinum analogs in murine bladder cancer

The activity of cis-diamminedichloroplatinum(II) was compared to two second generation platinum analogs, cis-diammine-1,1-cyclobutane dicarboxylate platinum(II) and cis-dichlorotransdihydroxybisisopropylamine platinum(IV) in the N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide-induced murine bladder tumor model and a tumor colony assay. Murine drug testing revealed that all three drugs were active against the MBT-2 tumor line, although cis-diamminedichloroplatinum(II) was more active than its analogs. All drugs produced enhanced inhibition of clonal growth with increasing drug exposure times. Cis-diamminedichloroplatinum(II) was more active against MBT-2 cells in the plateau growth phase versus the log growth phase after a one hour drug exposure. Similar differential activity depending upon the proliferative state of MBT-2 was not seen with the two platinum analogs. These two platinum analogs have somewhat less activity in vivo and in vitro than cis-diamminedichloroplatinum(II) and would be predicted to be less effective clinically in human bladder cancer than the parent compound.