Redistribution of rat renal allograft-responding leukocytes during rejection: 1. Model

We describe a model that enables us to trace the traffic of allograft-responding inflammatory leukocytes to and from the graft without handling of these cells in vitro. At different times after transplantation, the kidney transplant pedicle—including the artery, vein, and draining lymphatics—is clamped. The allograft-responding leukocytes are labeled by a [³H]thymidine pulse either in situ or in the systemic lymphoid organs of the recipient. Fifteen minutes later the pulse is chased with a 1000-fold excess of cold thymidine, and the clamp is opened. The animals are sacrificed 18 hr later, when a balance between the synthesis of new labeled leukocytes from the originally labeled ones and dilution of intracellular label has been achieved. This model was used to analyze the allograft-responding inflammatory cell traffic to and from a renal transplant performed across the major histocompatibility complex in the rat. A sizable traffic was observed to both directions: After systemic injection of label only 0.008 × 10⁶ labeled cells × hr^?1 were found to emigrate into a kidney allograft (control). Already on the third day after transplantation—when the in situ inflammatory response is still at its beginning—more than 0.3 × 10⁶ labeled cells × hr^?1 migrated from the host to the allograft and 1.6 × 10⁶ labeled cells × hr^?1 left the allograft to the recipient spleen. The first figure is several-fold higher than any previous estimate. The findings emphasize the systemic nature of the antiallograft inflammatory response.     

Mutually exclusive expression of fibronectin and glial fibrillary acidic protein in cultured brain cells

The reported expression of the cell surface-associated, mainly mesenchymal glycoprotein fibronectin by cultured glial cells is in discrepancy with recent work on brain tissue failing to demonstrate any glial or neuronal fibronectin. We have investigated the expression of fibronectin in relation to glial fibrillary acidic protein in cultured human glial and glioma cell lines as well as in cultures derived from newborn rat brain. Using double immunofluorescence technique we found that cells containing glial fibrillary acidic protein do not express fibronectin, and vice versa. The only exception to this rule was the occasional finding of fibronectin at points of cell-to-cell adhesion also in relation to cells containing glial fibrillary acidic protein. The results were also tested by polyacrylamide gel electrophoresis of the culture media of the human cell lines, and by subcultures from the brain of newborn rat, cultures stimulated with dibutyryl cyclic AMP (db-cAMP), and by vinblastine treatment of the cells. The lack of expression of fibronectin in cells containing glial fibrillary acidic protein, a gliospecific cytoskeletal protein, is discussed with reference to glio-mesenchymal interactions and glial markers in vitro.     

In situ effector pathways of allograft destruction. 3. Plasminogen activator activity in rat renal allografts

The question of which cell components in a rejecting rat renal allograft secrete plasminogen activator (PA) has been analyzed. Although normal renal parenchymal cells also secreted PA, most of the PA in a renal allograft (and to a lesser extent also in an autograft) was produced by the inflammatory leukocytes. Fractionation at 1 g demonstrated that the inflammatory cell population responsible for the PA production in the allograft sedimented together with the large mononuclear phagocytes (macrophages). Fractions purified for small blast cells and large lymphocytes did not contain any PA activity but they were able to induce resting peritoneal macrophages to produce PA when cocultured in vitro. The results demonstrate that the allograft-infiltrating mononuclear phagocytes are "activated" in the sense that they secrete PA and that the activation of mononuclear phagocytes at the site of inflammation may be partially regulated by the inflammatory lymphoid cells.     

Electron-dense endoplasmic reticulum-like profiles closely associated with mitochondria in glomus cells of the carotid body after fixation with oxalate

Fixation in the presence of oxalate was used to demonstrate the electron-dense Ca2+ precipitates in the endoplasmic reticulum in glomus cells of the carotid body. Glomus cells in intact carotid bodies or cells dissociated from the organ by treatment with collagenase were studied electron microscopically. In the intact organ as well as in dissociated glomus cells, electron-dense endoplasmic reticulum-like profiles were seen closely associated with mitochondria, while these lacked reaction product. The interspace between mitochondria was occupied by electron-dense, slightly distended ER, which appeared to contact the outer membrane of the mitochondria. Occasionally, a mitochondrion was in contact with several ER profiles or the ER formed an electron-dense 'cap' on the mitochondrion. The electron-dense precipitates could be removed from ultrathin sections with the calcium chelator ethyleneglycol-2(2-aminoethyl tetra-acetic acid) (EGTA). It is tentatively suggested that the endoplasmic reticulum could be involved in intracellular buffering of Ca2+ in the glomus cell, as has been previously suggested for neurons.     

Recognition of extracellular matrix components by neonatal and adult cardiac myocytes

The histogenesis of renal basement membranes was studied in grafts of avascular, 11-day-old mouse embryonic kidney rudiments grown on chick chorioallantoic membrane (CAM). Vessels of the chick CAM invade the mouse tissue during an incubation period of 7-10 days and eventually hybrid glomeruli composed of mouse epithelium and chick endothelium form. Formation of basement membranes during this development was followed by immunofluorescence and immunoperoxidase stainings using polyclonal and monoclonal antibodies against mouse and chick collagen type IV and against mouse laminin. These antibodies were species-specific as shown in immunochemical and immunohistologic analyses. The glomerular basement membrane contained both mouse and chick collagen type IV, demonstrating its dual cellular origin. All other basement membranes were either exclusively of chick origin (mesangium, vessels) or of mouse origin (tubuli, Bowman's capsule).     

Spinal cord dorsum potentials recorded in vivo after cold injury to the sural nerve in the rabbit

Evoked potentials were recorded in the spinal cord dorsum of rabbits during and after local cooling or freezing of the sural nerve. The potentials were elicited by stimulation through implanted bipolar electrodes distal and proximal to the site of cooling. Recordings were made with a unipolar electrode implanted dorsally into the epidural space.The first two negative deflections of the evoked field potentials (s- and n-potentials) decreased or disappeared during cooling to temperatures between 12 and 2 °C. Immediately following cooling the potentials were depressed by as much as 40% below that of controls. Gradual recovery of the nerve conduction velocity and of both potentials occurred between postoperative Days 20 and 60. Cooling of the nerve to between +2 and ?2 °C caused a 70–80% decrease in amplitude and the precooling values were not obtained within 90 days' follow-up. Local freezing of the sural nerve to ?45 °C resulted in disappearance of the cord dorsum potentials previously obtained by stimulation of the sural nerve with electrode distal to the site frozen. About 40% decrease occurred when stimulated proximal to the site frozen. A fast amplitude increase took place between days 50 and 100 and a slower increase between days 150 and 450 to values more than twice the preoperative amplitudes. A similar amplitude increase was obtained by stimulation of the nerve proximal to the site frozen.     

Factor VIII-related antigen : A pericellular matrix component of cultured human endothelial cells

We studied the extracellular localization of factor VIII-related antigen (VIIIR: Ag) in cultures of human endothelial cells. The cells deposited both VIIIR: Ag and fibronectin already during their initial adhesion phase and in immunofluorescence microscopy of spread cells extracellular VIIIR: Ag was localized to fibrils coaligning with pericellular fibronectin. When human fibroblasts, which do not synthesize VIIIR: Ag, were cultured in endothelial cell post-culture medium, a fibrillar matrix localization of VIIIR: Ag was seen, comparable to that of endothelial cell cultures. A fibrillar VIIIR: Ag-specific staining was also seen in cell-free pericellular matrices of endothelial cells, produced by deoxycholate treatment. In immunoelectron microscopy, VIIIR: Ag was seen in fibrillar extracellular material between and underneath the cells and in cell-free matrices of endothelial cells as well.In immunofluorescence microscopy of cell-free matrices, VIIIR: Ag codistributed with both fibronectin and type III procollagen. Digestion of the matrices with purified bacterial collagenase abolished the type III procollagen-specific fluorescence, whereas the fibrillar VIIIR: Ag-specific staining, codistributing with fibronectin, remained unaffected. In electrophoresis of isolated, metabolically labelled endothelial cell matrices, major polypeptides with M_r 220–240; 180; 160; 80 and 45 kD and some minor polypeptides were resolved. In addition, immunoblotting revealed fibronectin, VIIIR: Ag and type III procollagen as components of cell-free matrices of endothelial cells. Direct overlay of iodinated cellular fibronectin on electrophoretically separated polypeptides of cultured endothelial cells, transferred to nitrocellulose, suggested that fibronectin binds directly to VIIIR: Ag. Our results indicate that VIIIR: Ag produced by human endothelial cells is a component of the pericellular matrix and is not bound to collagen but may directly associate with fibronectin.     

The effect of the energy state of mitochondria on the kinetics of unidirectional cation fluxes

Unidirectional fluxes of triphenylmethylphosphonium and of Cs⁺ as its valinomycin complex were studied using trace concentrations of the cations. The rate constants of influx and efflux were estimated mainly at 0 °C from the uptake kinetics in respiring mitochondria and the in/out ratios in the steady state. The efflux rate constants in the energized state were also measured after dilution of the mitochondrial suspension in the steady state, and in deenergized mitochondria from the efflux rates of cations after inhibition of respiration. It was found that the energy state of mitochondria had little effect on the rate constants of efflux, while the rate of influx was strongly stimulated by respiration. The former finding is not readily explained by the classical chemiosmotic theory, since a transmembrane potential, negative on the inside, formed on energization would be expected to strongly inhibit the efflux of cations. The data may be explained by a pump-and-leak model in which localized electrical fields in hydrophobic domains of the membrane are coupled to the pumping of hydrophobic cations against an electrochemical gradient, while leaks would effect efflux.     

OKT4/8 ratio in the blood and in the graft during episodes of human renal allograft rejection

We have analyzed the frequency of T helper (Th) and T suppressor/killer (Ts/k) lymphocytes in the blood and in the renal allograft during episodes of rejection and during quiescence. Monoclonal OKT4 and OKT8 antibodies were used to mark the Th and Ts/k cells, respectively. Density centrifugation-separated mononuclear leukocytes and FACS IV cell sorter or the Staphylococcus aureus rosette assay were used to determine the ratio in the blood, with concordant results. Fine needle aspiration biopsy (FNAB) and the Staph. assay were used to demonstrate the lymphocyte subtypes in the graft. The mean OKT4/8 ratio in the blood was significantly lower in the transplant recipients than in healthy controls (1.1 +/- 0.7 vs 1.8 +/- 0.2, respectively, P = 0.000). The individual variation was, however, high and no correlation between the OKT4/8 ratio in the blood and the inflammatory episodes in situ was observed. During 19 of the 25 episodes of inflammation, the dominant lymphocyte subtype in the graft was the Ts/k cell. In the remaining six cases it was the Th cell. All rejection episodes of the former type were reversible, in the latter type, four out of six were irreversible.     

Lymphoid cell subclasses in rejecting renal allograft in the rat

We have quantitated the frequency of lymphoid cell subsets in rejecting renal allografts and in the spleen of the allograft recipient during drug-unmodified rejection in the rat. The number of inflammatory (white) cells in the graft was approximately similar to the number of white cells responding to the allograft in the recipient spleen. The inflammatory population of the graft consisted of lymphoid cells and mononuclear phagocytes, with increasing numbers of macrophages toward the end of rejection. Analysis of allograft cellular dispersates with monoclonal antibodies directed to the lymphoid cell subsets demonstrated that although the majority of allograft-infiltrating lymphocytes were T cells, a sizable B-cell proliferation and immunoglobulin synthesis was associated with the inflammatory response of rejection. Within the T-cell subset, the T suppressor/killer cells predominated in the graft whereas the predominant lymphoid cell subset responding to the allograft in the recipient spleen was the T helper cell.     

Immunohistochemical Characterization and Sensitivity to Human Adenovirus Serotypes 3, 5, and 11p of New Cell Lines Derived from Human Diffuse Grade II to IV Gliomas

BACKGROUND: Oncolytic adenoviruses show promise in targeting gliomas because they do not replicate in normal brain cells. However, clinical responses occur only in a subset of patients. One explanation could be the heterogenic expression level of virus receptors. Another contributing factor could be variable activity of tumor antiviral defenses in different glioma subtypes. METHODS: We established a collection of primary low-passage cell lines from different glioma subtypes (3 glioblastomas, 3 oligoastrocytomas, and 2 oligodendrogliomas) and assessed them for receptor expression and sensitivity to human adenovirus (HAd) serotypes 3, 5, and 11p. To gauge the impact of antiviral defenses, we also compared the infectivity of the oncolytic adenoviruses in interferon (IFN)-pretreated cells with IFN-sensitive Semliki Forest virus (SFV). RESULTS: Immunostaining revealed generally low expression of HAd5 receptor CAR in both primary tumors and derived cell lines. HAd11p receptor CD46 levels were maintained at moderate levels in both primary tumor samples and derived cell lines. HAd3 receptor DSG-2 was reduced in the cell lines compared to the tumors. Yet, at equal multiplicities of infection, the oncolytic potency of HAd5 in vitro in tumor-derived cells was comparable to HAd11p, whereas HAd3 lysed fewer cells than either of the other two HAd serotypes in 72 hours. IFN blocked replication of SFV, while HAds were rather unaffected. CONCLUSIONS: Adenovirus receptor levels on glioma-derived cell lines did not correlate with infection efficacy and may not be a relevant indicator of clinical oncolytic potency. Adenovirus receptor analysis should be preferentially performed on biopsies obtained perioperatively.     

Interferon affects the formation of adhesion plaques in human monocyte cultures

When monocytes isolated from human blood adhere to glass substratum, actin- and vinculin-containing punctate plaques rapidly appear at the ventral surface of the cells. We show here that highly purified human leukocyte interferon (IFN) can inhibit formation of these adhesion plaques in a dose-dependent manner. Complete inhibition was obtained when 300 IU/ml IFN were added into the cell-seeding medium. Plaques already formed in the absence of IFN were only partially affected by subsequent addition of IFN into the culture medium. Prevention by IFN of the formation of the adhesion plaques was associated with loosened attachment of the cells to the substratum. Effect of IFN on cellular morphology was complex. At higher doses, IFN added to the cultures within 24 h of seeding almost completely inhibited the differentiation of monocytes to macrophages and most of the cells remained rounded. At lower doses, however, an enhancement of the bipolar spreading was seen and the end result was a culture with predominantly elongated fibroblastoid cells. The latter cells, unlike the fibroblastoid cells in untreated monocyte-macrophage cultures, were completely devoid of the actin plaques, while the reorganization of vimentin-type intermediate filaments took place in a normal manner. These results further support the view that the actin- and vinculin-containing plaques have a role in mediating firm adherence of human monocytes to growth substratum.     

Differentiation and vascularization of the metanephric kidney grafted on the chorioallantoic membrane

The origin and development of mouse kidney vasculature were examined in chorioallantoic grafts of early kidney rudiments and of experimentally induced explants of separated metanephric mesenchymes. Whole kidney rudiments developed into advanced stages, expressed the segment-specific antigenic markers of tubules and the polyanionic coat of the glomeruli. In contrast to development in vitro, these grafts regularly showed glomeruli with an endothelial component and a basement membrane expressing type IV collagen and laminin. The glomerular endothelial cells in these grafts were shown to carry the nuclear structure of the host. This confirms the outside origin of these cells and the true hybrid nature of the glomeruli. When in vitro induced mesenchymes were grafted on chorioallantoic membranes, abundant vascular invasion was regularly found but properly vascularized glomeruli were exceptional. Uninduced, similarly grafted mesenchymal explants remained avascular as did the undifferentiated portions of partially induced mesenchymal blastemas. It is concluded that the stimulation of the host endothelial cells to invade into the differentiating mesenchyme requires the morphogenetic tissue interaction between the ureter bud and the mesenchyme. The induced metanephric cells presumably start to produce chemoattractants for endothelial cells at an early stage of differentiation. Kidney development thus seems to require an orderly, synchronized development of the three cell lineages: the branching ureter, the induced, tubule-forming mesenchyme, and the invading endothelial cells of outside origin.     

Response to Striefel and Glaros

In this response to the comments of Drs. Striefel and Glaros, we take issue with Dr. Striefel's assertion that the Declaration of Helsinki should not be regarded as a core ethical document, and demonstrate that his claim that the Declaration has no significance in or recognition by agencies of the United States Federal Government is in error. A reading of FDA and DHHS documents shows that the Declaration is not, as Dr. Striefel suggested, only an aspirational document, but also is clearly regarded as mandatory. Dr. Glaros' observations regarding the slippery concept of efficacy is very much on point, and we certainly agree with his call for more data. It is the means by which that can be accomplished that is problematic. We have suggested that the logic of placebo controls developed to render drug efficacy studies more rigorous and scientifically grounded does not translate well to many psychological and behavioral studies of efficacy. It is important for our discipline to rethink the standards by which new behavioral and psychological (including psychophysiological) interventions can be demonstrated to be efficacious. One possible approach that avoids ethical pitfalls and design impossibilities is the active control, which requires good demonstration of assay sensitivity. We hope that this discussion may stimulate further discussion about other approaches that do not depend upon the flawed placebo orthodoxy.     

Ethical analysis of cadaver supply and usage processes for research within the scope of the Helsinki Declaration

Recent technological developments have considerably transformed the supply, storage, and transportation processes of cadavers, creating new and previously unforeseen ethical challenges regarding cadaver usage. In this study, we analyzed two aspects of the cadaver processing system—cadaver supply and its use in research. Thereafter, we highlighted the major ethical concerns underlying these stages and correlated our search results with the ethical principles outlined in the Declaration of Helsinki (DoH), or Helsinki Declaration. To ensure the reliability and continuity of medical progress, human—especially cadaver—research depends on the ethical priorities as outlined in the DoH: respect for autonomy, privacy/confidentiality, risks/burdens/benefits, and the protection of vulnerable groups. According to our ethics analysis, which also corresponds with the ethics guidelines of the Consensus Panel on Research with the Recently Dead, the most ignored values were respect for autonomy and privacy/confidentiality issues. Based on these ethical concerns, we provide recommendations to address these challenges in anatomy research.     

Political legitimacy and research ethics

In democratic theory, “legitimacy” refers to the set of conditions that must be in place in order for the claims to authority of somebody to be deemed appropriate, and for their claims to compliance to be warranted. Though criteria of legitimacy have been elaborated in the context of democratic states, there is no reason for them not to be drawn up, with appropriate amendments, for other kinds of authority structures. This paper examines the claims to authority made over researchers by international bodies governing research ethics, who exercise their authority by the research ethics guidelines they produce (including recent revisions to the Declaration of Helsinki and CIOMS Guidelines). We argue that discussions of such bodies and sets of guidelines often elide questions of justification and questions of legitimacy, and that the grounds that might allow us to mount a strong case for the latter are at present sorely underdeveloped.     

Illegitimate authorship and flawed procedures: Fundamental,formal criticisms of the Declaration of Helsinki

Some of the recent criticisms published during and after the last revision process of the Declaration of Helsinki are directed at its basic legitimacy. In this article we want to have a closer look at the two criticisms we consider to be the most fundamental. The first criticism questions the legitimate authorship of the World Medical Association to publish a document such as the Declaration. The second fundamental criticism we want to examine argues that the last revision process failed to meet the standards for fair, democratic procedures. Although both criticisms deny the formal legitimacy of the Declaration in the most fundamental way, they have never been addressed in detail in a single article. We refute most of the related arguments. However, acknowledging some of the points made, improvements for future revision processes and versions of the Declaration of Helsinki are outlined.