Isolation of Hepatocytes and Stellate Cells from a Single Piece of Human Liver.

Co-transplantation of hepatocytes and hepatic stellate cells has been shown to increase the engraftment of transplanted hepatocytes in the liver. Here, we describe a method for the simultaneous isolation of human primary hepatocytes and hepatic stellate cells from the same donor for co-transplantation or for use in in vitro cell culture models.

Effects of intrauterine infusion of Trueperella pyogenes on endometrial mRNA expression of proinflammatory cytokines and luteolytic cascade genes and their association with luteal life span in dairy cows.

Objectives were to determine the effects of intrauterine infusion (IUI) of Trueperella pyogenes on endometrial expression of proinflammatory cytokines and luteal life span. Holstein cows (n = 32) were allocated randomly, in two replicates (15 then 17 cows), to receive one of three treatments on Day 5 of the estrous cycle: TP (n = 13), IUI containing 10(9) colony-forming units/mL of T. pyogenes; tumor necrosis factor (TNF; n = 9), IUI containing 1 µg of TNFα; and control (n = 10), IUI of saline solution. Five cows per treatment had uterine biopsies collected at 6, 12, and 24 hours after treatment to evaluate the endometrial messenger RNA expression of TNFα (TNF), interleukin-1ß (IL1B), IL6, IL8, prostaglandin E synthase (PGES), prostaglandin F synthase (PGFS), and oxytocin receptor (OXR), and histologic evidence of inflammation. Messenger RNA expression was measured using quantitative reverse transcription polymerase chain reaction. The remaining cows had ovaries scanned and blood collected for progesterone evaluation; however, only seven, four, and three cows in the TP, TNF, and control groups were used for comparison in replicate 2. The GLIMMIX procedure of SAS was used for statistical analysis. All TP and TNF cows had moderate to severe endometrial inflammation, whereas only one control had mild inflammation. Premature luteolysis occurred in three, one, and zero cows in the TP, TNF and control groups, respectively. Delayed luteolysis occurred in one TP and one TNF cow. Interleukin-1ß expression was greater in the TP cows than in the TNF cows at 24 hours after IUI. Moreover, IL6 expression tended to be greater for the TP cows than for the control cows at 12 hours after IUI. Interleukin 8 expression was greater in the TP cows than in the control and TNF cows at 24 hours after IUI. Oxytocin receptor expression tended to be greater for the TP cows and was greater for the TNF cows than for the control cows at 12 hours. The messenger RNA expressions of TNF, PGES, and PGFS were not affected by treatment, time, or their interaction. In conclusion, IUI of T. pyogenes or TNFα led to histologic evidence of inflammation and early luteolysis in some cows, which may have been caused by increased endometrial expression of proinflammatory cytokines (i.e., IL1B, IL6), chemokines (i.e., IL8), and luteolytic cascade factors (i.e., OXR).

An algorithm that predicts the viability and the yield of human hepatocytes isolated from remnant liver pieces obtained from liver resections.

Isolated human primary hepatocytes are an essential in vitro model for basic and clinical research. For successful application as a model, isolated hepatocytes need to have a good viability and be available in sufficient yield. Therefore, this study aims to identify donor characteristics, intra-operative factors, tissue processing and cell isolation parameters that affect the viability and yield of human hepatocytes. Remnant liver pieces from tissue designated as surgical waste were collected from 1034 donors with informed consent. Human hepatocytes were isolated by a two-step collagenase perfusion technique with modifications and hepatocyte yield and viability were subsequently determined. The accompanying patient data was collected and entered into a database. Univariate analyses found that the viability and the yield of hepatocytes were affected by many of the variables examined. Multivariate analyses were then carried out to confirm the factors that have a significant relationship with the viability and the yield. It was found that the viability of hepatocytes was significantly decreased by the presence of fibrosis, liver fat and with increasing gamma-glutamyltranspeptidase activity and bilirubin content. Yield was significantly decreased by the presence of liver fat, septal fibrosis, with increasing aspartate aminotransferase activity, cold ischemia times and weight of perfused liver. However, yield was significantly increased by chemotherapy treatment. In conclusion, this study determined the variables that have a significant effect on the viability and the yield of isolated human hepatocytes. These variables have been used to generate an algorithm that can calculate projected viability and yield of isolated human hepatocytes. In this way, projected viability can be determined even before isolation of hepatocytes, so that donors that result in high viability and yield can be identified. Further, if the viability and yield of the isolated hepatocytes is lower than expected, this will highlight a methodological problem that can be addressed.

Biobanking for research in surgery: are surgeons in charge for advancing translational research or mere assistants in biomaterial and data preservation?

BACKGROUND: High-quality biospecimens of human origin with annotated clinical and procedural data are an important tool for biomedical research, not only to map physiology, pathophysiology and aetiology but also to go beyond in translational research. This has opened a new special field of research known as 'biobanking', which focuses on how to collect, store and provide these specimens and data, and which is substantially supported by national and European funding. PURPOSE: An overview on biobanking is given, with a closer look on a clinical setting, concerning a necessary distinction from clinical trials and studies as well as a comparison of prospective sample collection with secondary use of archived samples from diagnostics. Based on a summary of possible use and scientific impact of human tissue in research, it is shown how surgical expertise boosts the scientific value of specimens and data. Finally, an assessment of legal and ethical issues especially from a surgical perspective is given, followed by a model of interdisciplinary biobanking within a joint 'centre' that as synergistic structure merges essential input from surgery as well as laboratory medicine, pathology and biometry. CONCLUSION: Within the domain of biobanking, surgeons have to develop a better awareness of their role within translational research, not only on the level of medical faculties but also as nationally and internationally funded initiatives. Therefore, the authors suggest a platform for biobanking within the German association of surgeons in analogy to the existing special interest group for clinical trials.

RNA stability in human liver: comparison of different processing times, temperatures and methods.

The accuracy of information garnered by real-time quantitative polymerase chain reaction (RT-qPCR), an important technology for elucidating molecular mechanisms of disease, is dependent on tissue quality. Thus, this study aimed to determine the effects of intra-operative manipulation, extended processing times, different temperatures or storage in RNAlater on RNA quality in liver samples for tissue banking. Liver samples, flash-frozen or in RNAlater, were collected over a time course (during surgery before blood arrest up to 1 day after surgery) with samples kept either at room temperature (RT) or on ice. This study showed that at the longest time-point at RT, the RNA quality decreased significantly by 20%. However, relative gene expressions of FOS, GUSB, MYC, HIF1α and GFER were in general not significantly different when the time-points were compared. In conclusion, samples should be kept on ice during processing, and either RNAlater or snap-freezing should be utilised for storage. Further, intra-operative manipulation and extended postoperative processing time generally does not change relative gene expression levels for the 5 genes studied, making such sampling suitable for RT-qPCR analysis. Thus, if relative gene expression of a gene of interest is stable, these guidelines will lead to increased accrual of samples to the tissue bank.

Enhanced alveolar bone loss in a model of non-invasive periodontitis in rice rats.

OBJECTIVE: The rice rat (Oryzomys palustris) develops periodontitis-like lesions when fed a diet rich in sucrose and casein (H-SC). We aimed to establish whether this model can accurately mimic the development of human periodontitis. MATERIALS AND METHODS: For this purpose, 28-day-old rice rats (15/group) were assigned to standard (STD) or H-SC diets and sacrificed after 6, 12, and 18 weeks. Jaws were processed for morphometric, histometric, histologic, histomorphometric, and micro-CT analyses. RESULTS: We found a progressive increase in horizontal alveolar bone loss (ABL) with age in maxillae of rats fed the STD diet as determined by morphometry. The H-SC diet exacerbated horizontal ABL at the palatal surface at 12 and 18 weeks. Furthermore, increased vertical ABL was detected in mandibles and maxillae of rats fed the H-SC diet for 12 and/or 18 weeks by histometry and micro-CT. Remarkably, the H-SC diet significantly increased bone remodeling at the interproximal alveolar bone of mandibles from rats fed for 6 weeks, but not in those fed for longer periods. CONCLUSIONS: These findings indicate that the H-SC diet induced a transient increase in alveolar bone remodeling, which is followed by ABL characteristic of moderate periodontitis.

Impact of pneumoperitoneum on trocar site implantation of colon cancer in hamster model.

BACKGROUND: Numerous anecdotal reports have documented the spread of colon cancer to trocar sites after laparoscopic-assisted colectomy. We hypothesized that the pneumoperitoneum of laparoscopy potentiated tumor adherence to trocar sites. PURPOSE: This study was designed to determine the effect of CO2 pneumoperitoneum on the rate of tumor implantation at trocar sites. METHODS: Viable GW-39 human colon cancer cells were injected into the abdominal cavity of hamsters (2 x 10(6) cells/hamster). A midline laparotomy, insertion of four 5-mm trocars, injection of viable cells into the mesentery of the cecum, and free peritoneal cavity was performed in two groups: one control group (41) who did not receive a pneumoperitoneum and a comparison group (50) who underwent pneumoperitoneum for ten minutes at an insufflation pressure of 10 mmHg. Animals were killed at six weeks, and hematoxylin and eosin-stained sections of trocar wounds, midline wound, small intestine, cecum, liver, and lung were examined by a veterinary pathologist, who was blinded to operation. RESULTS: Pneumoperitoneum increased tumor implantation in the cecal mesentery and the midline incision (P < 0.05) but did not effect recurrence in the liver, lung, or jejunum. Trocar site implantation tripled with the addition of pneumoperitoneum increased implantation of pneumoperitoneum (26 vs. 75 percent) (P < 0.0001). CONCLUSION: Pneumoperitoneum increased implantation of free intra-abdominal cancer cells at wound sites on the abdominal wall or within the abdominal cavity in this animal model. The use of pneumoperitoneum during laparoscopy in patients with colon cancer should only be performed in a protocol setting to evaluate the effect of pneumoperitoneum on the treatment of cancer.

Effects of polyaspartic acid on pharmacokinetics of tobramycin in two strains of rat.

To provide insight into polyaspartic acid nephroprotection and differences in aminoglycoside renal toxicity between two rat strains, the single-dose pharmacokinetics of tobramycin was examined in the presence and absence of polyaspartic acid. Following a single subcutaneous 6.5-mg/kg dose of tobramycin alone, higher aminoglycoside concentrations were measured in Sprague-Dawley rats than in Fischer rats (P < 0.05). Simultaneous administration of polyaspartic acid (50 mg/kg) and tobramycin did not alter the concentrations of tobramycin in serum. The amount of tobramycin in renal tissue and the amount recovered in urine over a 24-h period were greater in both rat strains when tobramycin and polyaspartic acid were given concomitantly. In summary, polyaspartic acid did not alter the concentrations in serum achieved after a single dose of tobramycin in two different rat strains but did result in higher renal concentrations and greater urinary excretion of tobramycin.

Lack of in vivo evidence of a cytochrome P450 metabolite participating in aminoglycoside nephrotoxicity.

Recent in vitro evidence has suggested that the cytotoxicity of aminoglycosides may be mediated by a metabolite generated by the hepatic cytochrome P450 drug-metabolizing system. This postulate has been tested by pretreating rats with cobalt protoporphyrin IX (CoP) to suppress hepatic P450 levels prior to administration of gentamicin. CoP pretreatment was observed to suppress antipyrine clearance markedly but not to alter gentamicin nephrotoxicity.

Differences in the sensitivity of Fischer and Sprague-Dawley rats to aminoglycoside nephrotoxicity.

Because of the anecdotal but unconfirmed inferences of the greater sensitivity of Fischer rats to aminoglycoside nephrotoxicity, an intrastudy comparison of the sensitivity of Fischer and Sprague-Dawley rats to aminoglycoside nephrotoxicity was undertaken. Tobramycin was administered at 3 dose levels to each strain of rat for 10 days. Nephrotoxicity was evaluated utilizing a spectrum of both functional and morphologic assessments of renal proximal tubular integrity. The results confirm that the aged matched Fischer rat is more sensitive to the nephrotoxic effect of tobramycin than the Sprague-Dawley. These results also suggest an opportunity to study quantitative and perhaps qualitative differences in pathogenic mechanisms of aminoglycoside nephrotoxicity in a single laboratory animal species.

Alterations in lung prostaglandin synthesis and release in murine respiratory mycoplasmosis.

Pathogenetic mechanisms in murine respiratory mycoplasmosis are poorly understood; however, non-specific immune responses appear to be important in controlling the growth of Mycoplasma pulmonis in vitro. To date, no study has examined the role of pulmonary prostaglandin production during the development of M. pulmonis infection. The present study was designed to determine if alterations in pulmonary prostaglandin synthesis and release occur in M. pulmonis infection and the possible role for prostaglandins in the modulation/pathogenesis of murine respiratory mycoplasmosis. Ten to 20 days after intranasal inoculation of pathogen-fee F344 rats with M. pulmonis, lung lavage concentrations of prostaglandin E (PGE) and thromboxane A2 (TxA2) were significantly elevated. To confirm a role for prostaglandins in the pathogenesis of murine mycoplasmosis we blocked the cyclo-oxygenase pathway with indomethacin. Indomethacin-treated rats had significantly lower lavage levels of PGE and TxA2 and significantly increased numbers of M. pulmonis in the lung. These data indicate that prostaglandins may be involved in the pathogenesis of murine respiratory mycoplasmosis, possibly through alteration of mycoplasmacidal and/or mycoplasmastatic mechanisms.

Inapparent Streptococcus pneumoniae type 35 infections in commercial rats and mice.

Streptococcus pneumoniae was isolated from specific-pathogen-free rodents in two rooms at a commercial breeding facility during vendor surveillance testing. In a survey of 274 animals from the two rooms over a period of 7 months, capsular serotype 35 S. pneumoniae was isolated from the upper respiratory tracts of 11% (9 of 82) of C57BL/6 mice in room A and 14% (10 of 72) of F344 rats in room B, but not from WKY rats, BALB/c mice or DBA/2 mice from room A. In both C57BL/6 mice and F344 rats, older rodents had higher colonization frequencies. Nasal lavage cultures gave the best results in identifying colonized rodents. No clinical illness or microscopic lesions were associated with pneumococcal colonization in rats or mice, and no other evidence of potential pathogen infection was found except for positive serologic tests for mouse rotavirus in mice. This is the first report of natural pneumococcal infection in mice, and the first report of type 35 S. pneumoniae infection in rodents. The findings support an earlier observation that pneumococcal infections in rat colonies tend to be monotypic and suggest that the same may be true in mice.

Molluscum contagiosum in a horse with granulomatous enteritis.

Widespread cutaneous papules in a yearling Standardbred filly were attributed by light and electron microscopic examination to molluscum contagiosum. Concomitant granulomatous enteritis, suspected clinically due to protein-losing enteropathy, was verified histopathologically. An associated altered altered immune response is suggested as the reason for the widespread poxvirus infection.

Verminous (Strongylus vulgaris) myelitis in a donkey.

A fifth stage Strongylus vulgaris migrated through the spinal cord of a 2-year-old, male donkey resulting in progressive paraparesis and then tetraplegia. A profound neutrophilic pleocytosis was detected on analysis of cerebrospinal fluid. The parasite appeared to have entered the mid-lumbar spinal cord, migrated to the cranial thoracic segments, exited, then re-entered the spinal cord a few segments craniad. It then traveled further cranially and was found in the third cervical spinal cord segment. Some parts of the lesion were remarkably free from tissue necrosis, hemorrhage and inflammation. Severe granulomatous myelitis with hemorrhage and necrosis were seen at other sites. The latter were quite similar to lesions seen in equine protozoal myeloencephalitis.