Shift of C3 deposition from localization in the glomerulus into the tubulo-interstitial compartment in the absence of secreted IgM in immune complex glomerulonephritis.

The role of secretory IgM in protecting kidney tissue from immune complex glomerulonephritis induced by 4 mg horse spleen apoferritin and 0.05 mg lipopolysaccharide has been investigated in mutant mice in which B cells do not secrete IgM, but are capable of expressing surface IgM and IgD and secreting other Ig isotypes. Glomerular size, number of glomeruli per cross-section, glomerular cellularity and urine content of protein and creatinine was comparable in treated secreted IgM (sIgM)-deficient and wild-type mice. Assessment of urinary proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a 30 kDa low molecular weight protein in treated sIgM-deficient animals only, reflecting dysfunction of proximal tubules. A shift of bound C3 from glomeruli to the tubulo-interstitial compartment in sIgM-deficient mice also suggests tubulo-interstitial damage. In contrast, local C3 synthesis within the kidney tissue did not differ between the two treated groups. Apoptosis physiologically present to maintain kidney cell homeostasis was increased slightly in treated wild-type mice. These results indicate that secretory IgM can protect the tubulo-interstitial compartment from immune complex-induced damage without having an effect on the glomerulus.

Serum free cell culture: the free access online database.

The cultivation of cells in vitro is an important tool for biomedical research and production purposes. The supplementation of animal/human cell culture media with sera (components) of animal origin remains still standard, providing for e.g. necessary nutrition, shear protection, growth factors and cytokines. Because of undefined composition, risk of contaminations, the cost factor and also animal welfare considerations concerning the production of sera, the conversion to serum free alternatives is promoted by regulatory authorities, industry and the research community in general. To support this trend and to help save one of the scientists most valuable resources -- time -- a data bank was compiled of commercially available formulations, searchable for products, applications, cell lines and manufacturers. The database is accessible free of charge in HTML format and as PDF download, the informations are checked and updated twice a year. Problems concerning serum free cell culture are discussed at and comments are welcome.

Proliferation of myocytes on different ECM-components of animal/human origin in vitro.

For the purpose of tissue regeneration gels of reconstituted basement membrane have been suggested as a vehicle to transfer autologous cells. Results do look promising, but it should be considered that extracellular matrix (ECM) gel (Matrigel) is a soluble extract of Engelbreth-Holm-Swarm (EHS) mouse tumor. Therefore objections arising concerning possible risks complicate clinical use in human subjects. Aim of this study was to determine whether ECM-components of human origin can be used as substitutes for tissue engineering tasks as proposed previously. Proliferation capability and viability of primary rat myocytes and rat myocyte cell lines were determined on days 1, 2, 4 and 8 after inocculation of the cells. Pooled data suggest that an appropriate combination of human ECM and human Collagen Type IV may represent an approach with good prospects.

[Concerning cell cultures for biocompatibility-testing: monitoring by DNA-fingerprinting]. / Ad Zellkulturen zur Biomaterialuntersuchung: Uberwachung mit DNA-Fingerabdrücken.

Cell cultures are innovative tools for e.g. biocompatibility testing of biomaterials in vitro. In our studies we used fibroblast, endothelial cell and chondrocyte cultures of human origin and of the test animal species most common for this purpose in vivo. Verification of the identity of these cells is obligatory for reproducibility of the tests and valid interpretation of the results. Cultured cells have to be checked for identity, contaminations of various origins and also for genomic mutations occuring during prolonged cultivation in vitro or due to exposition to biomaterials. Furthermore, the risk of genetic cross-contamination with other cells increases with the number of cell cultures passaged parallel in the same laboratory. Therefore, we generated reference fingerprints of the cultures in varying passages for comparative monitoring of cells purposed for in vitro tests. Minisattelite DNA polymorhism resulting in reproducible individual DNA fingerprints is very discriminatory and can be used for cell culture monitoring. The patterns are stable over several passages, although sudden changes did happen in two cases, i.e. loss/gain of bands or changes in band-intensity, indicating massive genomic mutations of the cultures in vitro. Influences of biomaterials on the prints could not be detected. Several tasks can be followed at the same time: detection of contaminant cells, identification of these cells of primary culture origin used for in vitro testing and finally, monitoring for eventual genomic mutations due to prolonged cultivation or contact to biomaterials. Inconclusive results in just one of these aspects should lead to the disqualification of the monitored cultures from usage in vitro.

The role of intra-abdominal pressure on splanchnic and pulmonary hemodynamic and metabolic changes during carbon dioxide pneumoperitoneum.

BACKGROUND AND METHODS: To find an intra-abdominal pressure (IAP) range for laparoscopic procedures that elicits only moderate splanchnic and pulmonary hemodynamic and metabolic changes, including hepatic and intestinal tissue pH and superficial hepatic blood flow, we installed an IAP of 7 and 14 mm Hg each for 30 minutes in 10 healthy pigs (30 +/- 4 kg). RESULTS: In parallel with the increase of IAP, the mean transmural pulmonary artery pressure increased (from 25 +/- 3 to 27 +/- 4 at 7 mm Hg IAP and 30 +/- 6 mm Hg at 14 mm Hg IAP, p < 0.05); the pulmonary artery-to-pulmonary capillary wedge pressure gradient also increased (from 17 +/- 2.7 to 21 +/- 3 mm Hg at 7 mm Hg IAP and 24 +/- 4.2 mm Hg at 14 mm Hg IAP, p < 0.01), and the arterial oxygenation decreased (p < 0.005). Relevant changes at an IAP of 14 mm Hg were observed in right atrial pressure during inspiration (from 7 +/- 2 to 12 +/- 3 mm Hg, p < 0. 0001) and in abdominal aortic flow (from 1.43 +/- 0.4 to 1.19 +/- 0. 3 L/min, p < 0.01). However, transmural right atrial pressure and cardiac output remained essentially unchanged. Portal and hepatic venous pressure increased in parallel with the IAP (portal: from 12 +/- 3 to 17 +/- 3 at 7 mm Hg IAP and 22 +/- 3 mm Hg at 14 mm Hg IAP, p < 0.01; hepatic venous: from 8 +/- 3 to 14 +/- 6 at 7 mm Hg IAP and 19 +/- 6 mm Hg at 14 mm Hg IAP, p < 0.005), but the transmural portal and hepatic venous pressures decreased (p < 0.01), indicating decreased venous filling. Portal flow was maintained at 7 mm Hg but decreased at 14 mm Hg from 474 +/- 199 to 395 +/- 175 mL/min (p < 0. 01), whereas hepatic arterial flow remained stable. Hepatic superficial blood flow decreased during insufflation and increased after desufflation. Tissue pH fell together with portal and hepatic venous pH (intestinal: from 7.323 +/- 0.05 to 7.217 +/- 0.04; hepatic: from 7.259 +/- 0.04 to 7.125 +/- 0.06, both p < 0.01) at 14 mm Hg. CONCLUSION: The hemodynamic and metabolic derangement in the pulmonary and splanchnic compartments are dependent on the extent of carbon dioxide pneumoperitoneum. The effect of low IAP (7 mm Hg) on splanchnic perfusion is minimal. However, higher IAPs (14 mm Hg) decrease portal and superficial hepatic blood flow and hepatic and intestinal tissue pH.

Improved laparoscopic operating techniques using a digital enhancement video system.

We conducted a prospective, randomized trial in a piglet model to assess the advantages of a digital enhancement video system during difficult endoscopic procedures. Laparoscopic pelvic lymphadenectomy was performed with either conventional two-dimensional or digitally enhanced videoendoscopy. With equal amounts of extirpated lymph nodes, the operations performed with digitally enhanced videoendoscopy were significantly shorter (left lymphadenectomy p = 0.0001, right lymphadenectomy p = 0.001), had less blood loss and fewer false movements, and were associated with no complications, compared with those performed with a two-dimensional videosystem. Thus the accuracy of difficult laparoscopic techniques can be improved by three-dimensional visual perception.

Gene therapy in surgery: Part II: Application to septic shock and to organ transplantation.

BACKGROUND: With the increasing body of knowledge in molecular biology, gene transfer respectively gene therapy becomes more and more a valid therapeutic option. METHODS: This is a critical review of gene therapy protocols for treatment of different types of cancer. Furthermore, the pathophysiological mechanism, therapeutically strategies as well as experimental approaches toward gene transfer in septic shock and organ transplantation are critically elucidated. RESULTS: Gene transfer as a therapeutic option was first successfully applied in children with severe combined immunodeficiency (SCID) in 1990. The majority of gene marking or gene therapy protocols approved for human clinical trials to date are related to the treatment of cancer. Besides viral vectors for brain tumors, non-viral vectors, liposomes particularly, with almost no side effects are increasingly used. CONCLUSIONS: Different approaches of gene transfer in cancer patients are under investigation. Experimental data of septic shock treatment and rejection therapy of the allograft in organ recipients with gene transfer are encouraging for future applications in clinical trials.

[Circulatory support by an electrically stimulated muscle flap. Experimental experiences]. / Kreislaufunterstützung durch elektrisch stimulierte Muskellappen. Experimentelle Erfahrungen.

Functional electrical stimulation of the latissimus dorsi muscle flap for circulatory assistance extends the traditional concept of using this flap for reconstructive procedures into the field of cardiac surgery. It requires a transformed muscle which is able to contract for long periods of time without fatigue. Two main groups of experiments have been carried out in sheep. In six sheep the latissimus dorsi muscle (MLD) was transformed into a fatigue-resistant muscle by the means of multichannel stimulation of the supplying motor nerve. After that, stimulation of MLD at a frequency of 70 contractions per minute could be performed continuously without significant muscle fatigue. The loss of maximal force caused by the conditioning procedure was about one third of the initial force. In a second series of acute experiments the MLD was used for cardiomyoplasty. The muscle was divided into two parts which were wrapped around the heart in two different forms. The resting tension of the muscle was preserved. EKG-synchronous stimulation resulted in an increase in left ventricular pressure between 12 and 53%. The increase in arterial pressure was between 10,6 and 58%.

A physical analog of the failing left ventricle for in vitro studies of mechanical wall actuation.

Mechanical repowering of a failing heart with devices or skeletal muscle could circumvent blood-pump lining problems. Requirements are complex: indefinite support with preservation of valve competence and coronary flow, avoidance of wall coaptation, and allowance of both rapid low impedance refilling and independent left and right pressures. An accurate in vitro physical failing-heart analog could facilitate the choice and screening of surgical and engineering approaches in mock circulation experiments. Prosthetic models, transplant recipient hearts, normal animal hearts, existing in vivo animal failure models, and failing cadaver hearts all have serious limitations. One hundred and four excised porcine hearts were dilated and fixed by three iterative protocols. Geometric and passive mechanical parameters were assessed and compared with targets expected for an end-stage failing heart. For Protocol 3, Subgroup 2 (reinforcing valve support, dilatation by compliant ventricular balloon, and ethyl alcohol fixation), the left ventricular shape and capacity (ellipsoid, 201-377 ml/500 g of heart weight), passive valve function, wall flexural rigidity (Et3 range 0.101-0.331 Nm), and refilling mechanics (99 +/- 17.46 ml during 200-400 ms at < or = 10 mm Hg transmural gradient) were all within goal criteria.

Influence of different conditioning methods on force and fatigue resistance in chronically stimulated skeletal muscles.

A study was undertaken to compare different conditioning methods for the transformation of latissimus dorsi muscle into a fatigue resistant one for application in circulatory assist. In ten sheep four electrodes were sutured to the epineurium of the left thoracodorsal nerve for indirect electrical stimulation of the latissimus dorsi muscle. In six sheep a "carousel stimulation," a special multichannel stimulation, in combination with a recently developed conditioning protocol was used for muscle conditioning (multichannel method). In four sheep, a conventional stimulation protocol using single channel stimulation was applied for transformation of the muscle (single channel method). The final experiments were carried out when fatigue resistance was obtained. The maximum tetanic forces at different preloads were determined and fatigue resistance was tested during 20 minutes of continuous stimulation. Both conditioning patterns led to fatigue-free chronic stimulation. Muscles conditioned by multichannel stimulation exhibited between 20% and 33% less force than the contralateral unconditioned muscles, whereas in the single channel group this loss was between 32% and 43%. Thus, the multichannel method revealed relatively superior in preserving muscle force for chronic stimulation.

Long-term ventricular wall actuation: can and should it be systematically explored?

Hearts fail because myocardial power fails. Assist, support, or replacement devices fail, at least in part, because their blood-contacting surfaces fail. Mechanical repowering of a failing heart might circumvent these difficulties by preserving a largely healthy endocardium while correcting the basic deficit, power. Any serious consideration of doing this though must confront some difficult requirements. Effective indefinite support must be coupled with preservation or restoration of valve competence, coronary flow, rapid low-impedance refilling and independent left and right pressures; the avoidance of wall coaptation; hardware that fits in the available space; and unless muscle powered, adaptability to a deliverable form of power. Despite earlier intense interest in acute mechanical devices and later empiric study of muscle wraps, little systematic methodical work has been done on elucidating and meeting these practical requirements. Concerted efforts toward developing research tools and techniques for their study and then finding mechanisms to meet them could well yield one or more effective modalities that circumvent a major obstacle to the indefinite mechanical treatment of heart failure.

Experimental in situ conditioning of the latissimus dorsi muscle for circulatory assist by multichannel stimulation.

This study was undertaken to survey the changes in force and fatigue of the latissimus dorsi muscle during transformation into a fatigue-resistant muscle by indirect or nervous multichannel stimulation. In sheep, a silicone chamber connected to a pressure-transducing system was implanted under the left latissimus dorsi muscle. Muscle conditioning was performed by multichannel (carousel) stimulation of the thoracodorsal nerve. The program was started with active periods of 10 min/h producing 10 tetanic contractions/min. It was increased until 70 contractions/min could be performed during 24 h. The changes of muscle force and fatigue were monitored by the silicone balloon system. After a mean period of 22 weeks, fatigue resistance was reached. The fatigue-resistant muscle was able to produce a pressure of about 100 mm Hg in the balloon. After finishing the conditioning procedure, muscle forces and the fatigue resistance of the conditioned muscle were evaluated. The conditioned muscle showed only a minimal decrease of force during 20 min. Under a preload of 20 N, it exhibited a maximum tetanic tension of 95 N.

Action of endogenous atrial natriuretic peptide in calves with experimental acute central venous congestion and low cardiac output.

STUDY OBJECTIVE: The aim of the study was to investigate plasma concentrations of atrial natriuretic peptide, aldosterone, and renin during experimentally induced acute central venous congestion. DESIGN: Two experimental calf models were used: (1) right heart failure due to pulmonary artery obstruction; (2) inferior vena cava syndrome produced by inferior vena caval obstruction. Hormonal responses and haemodynamic variables were measured over 6 h. SUBJECTS: Experiments were performed on three female "Schwarzbund" calves, age 3 months, weight 92 +/- 8 kg. MEASUREMENTS AND MAIN RESULTS: In the pulmonary artery obstructed group there was an increase of plasma aldosterone from 6.5(SEM 1.6) to 22.1(3.2) ng.dl-1 (p less than 0.05), of renin from 0.7(0.1) to 2.5(0.3) Goldblatt units x 10(-4).ml-1 (p less than 0.05), and of atrial natriuretic peptide from 22.1(4.5) to 141.4(27.8) pmol.litre-1 (p less than 0.05). During inferior vena caval obstruction, aldosterone increased from 2.4(0.4) to 20.9(2.0) ng.dl-1 (p less than 0.05), and renin increased from 0.4(0.05) to 2.0(0.20) Goldblatt units x 10(-4).ml-1 (p less than 0.05). In this experiment, atrial natriuretic peptide remained unchanged. Cardiac output decreased in both groups. There was significant fluid and electrolyte retention during both experiments, with urine volume decreasing from 87.7(11.6) to 35.0(1.2) ml-h-1 in experiment (1), and from 185(14) to 95.7(8.6) ml.h-1 in experiment (2). CONCLUSIONS: The study suggests (1) that in an experimental acute state of reduced cardiac output due to pulmonary artery stenosis with constantly increased right heart pressures, raised endogenous atrial natriuretic peptide failed to induce diuresis and natriuresis; (2) that in acute right heart failure, renin and aldosterone secretion could not be suppressed by raised atrial natriuretic peptide concentrations; and (3) atrial natriuretic peptide secretion seemed to be exhausted after 6 h continuous atrial distension.

Atrial natriuretic factor production and secretion during clinical total artificial heart-bridge-to-transplantation.

In 5 total artificial heart (TAH)-bridge-to-transplant (BTT) patients production and secretion of atrial natriuretic factor (ANF)1 was studied before, during, and after TAH implantation. Bridging periods lasted between 9 and 28 days. Atrial biopsies were taken during implantation, and after TAH explantation for histologic and histochemical investigations to evaluate differences in morphology and ANF-content of the specific atrial granules. Plasma concentrations of ANF (pANF), aldosterone (PAC) and renin (PRC) were measured daily, as were hemodynamic parameters. In the preoperative state, pANF was always markedly elevated, while during TAH bridging, pANF remained moderately elevated with fluctuations. A positive correlation between pANF levels and right atrial pressure (RAP) was seen in all patients (p less than 0.05). Slight correlation was also observed between pANF and left atrial pressure (LAP), but no correlation was seen between pANF and systemic blood pressure, and no consistent pattern was seen in the plasma concentrations of either renin or aldosterone. After heart transplantation (HTX), which was performed in 3 patients, pANF levels were significantly higher than during TAH, and continued to show a positive correlation with RAP. From our data, we conclude that ANF production sites and secretory mechanisms remain intact during TAH-bridging, although upon implantation of a TAH, the remaining atria are deprived of all coronary blood supply and most autonomic innervation.