Synergy of local, regional, and systemic non-specific stressors for host defense against pathogens.

The immune brinksmanship conceptual model postulates that many of the non-specific stressful components of the acute-phase response (e.g. fever, loss of appetite, iron and zinc sequestration) are host-derived systemic stressors used with the "hope" that pathogens will be harmed relatively more than the host. The concept proposes that pathogens, needing to grow and replicate in order to invade their host, should be relatively more vulnerable to non-specific systemic stress than the host and its cells. However, the conceptual model acknowledges the risk to the host in that the gamble to induce systemic self-harming stress to harm pathogens may not pay off in the end. We developed an agent-based model of a simplified host having a local infection to evaluate the utility of non-specific stress, harming host and pathogen alike, for host defense. With our model, we explore the benefits and risks of self-harming strategies and confirm the immune brinksmanship concept of the potential of systemic stressors to be an effective but costly host defense. Further, we extend the concept by including in our model the effects of local and regional non-specific stressors at sites of infection as additional defenses. These include the locally hostile inflammatory environment and the stress of reduced perfusion in the infected region due to coagulation and vascular leakage. In our model, we found that completely non-specific stressors at the local, regional, and systemic levels can act synergistically in host defense.

P53+/- hemizygous knockout mouse: overview of available data.

The performance of the p53-/- transgenic (knockout) mouse model was evaluated through review of the data from 31 short-term carcinogenicity studies with 21 compounds tested as part of the International Life Sciences Institute's (ILSI) Alternatives to Carcinogenicity Testing (ACT) project, together with data from other studies which used comparable protocols. As expected based on the hypothesis for the model, a significant number (12/16 or 75%) of the genotoxic human and/or rodent carcinogens tested were positive and the positive control, p-cresidine, gave reproducible responses across laboratories (18/19 studies positive in bladder). An immunosuppressive human carcinogen, cyclosporin A, was positive for lymphomas but produced a similar response in wild type mice. Two hormones that are human tumorigens, diethylstilbestrol and 17beta-estradiol, gave positive and equivocal results, respectively, in the pituitary with p53-deficient mice showing a greater incidence of proliferative lesions than wild type. None of the 22 nongenotoxic rodent carcinogens that have been tested produced a positive response but 2 compounds in this category, chloroform and diethylhexylphthalate, were judged equivocal based on effects in liver and kidney respectively. Four genotoxic noncarcinogens and 6 nongenotoxic, noncarcinogens were also negative. In total (excluding compounds with equivocal results), 42 of 48 compounds or 88% gave results that were concordant with expectations. The technical lessons learned from the ILSI ACT-sponsored testing in the p53+/- model are discussed.

Genetic conflict and apoptosis.

The benefits of apoptosis in the removal of unnecessary, damaged, or dangerous cells are dependent on the altruism resulting from the absence of genetic conflict between genes in cells. However, this altruism can be exploited by self-promoting or ultra-selfish genes. These self-promoting genes can be endogenous, as with neoplasia or germ cell mutations, or exogenous, as with cellular pathogens. The fundamental flaw of apoptosis is that its development and maintenance as a system is constantly opposed by the emergence of self-promoting genes. Since apoptotically impaired cells cannot be relied on to kill themselves, apoptotic input from other cells is required for controlling self-promoting genes. Certain unique features of germ cell development, such as linkage by cytoplasmic bridges and the requirement for granulosa or Sertoli cells, appear to serve this requirement for control of self-promoting genes.

Implications of early apoptosis of infected cells as an important host defense.

Apoptosis is widely recognized as being a host defense against viral infections, since viruses require live cells. There has been increasing acceptance of the view that apoptosis is also a defense against other intracellular pathogens and even against pathogens that adhere to host cells. An implication of apoptosis being a host defense is a need to reassess to what extent the cell death at infection sites may constitute a protective host response. A concept stressed here is that infected cells are a hazard to other cells and to the individual, so the benefits of early apoptosis are emphasized. Therefore, promoting the survival of infected cells, even though still functional, may carry risks. A further consideration is the possibility that the apoptotic stimulus of nutrient restriction may be acting in infection-induced anorexia to promote apoptosis of infected cells, thereby serving as a non-specific host defense.

Why infection-induced anorexia? The case for enhanced apoptosis of infected cells.

A medically important paradox is why the body's own cytokines lead to reduced appetite and apparently inefficient metabolism as part of the acute-phase response. This self-induced nutrient restriction occurs just when the body must maintain a fever and other defensive functions. This paradox is often ignored or considered a metabolic derangement. Others, recognizing it to be a programmed response which must have net beneficial effects, consider the nutrient restriction to be an attempt to deny resources to infectious organisms. However, this explanation fails to address how the pathogen can be harmed more than the host. The hypothesis presented here offers an explanation. Apoptosis, or cell suicide, is becoming recognized as a useful defense against intracellular parasites, and nutrient restriction promotes apoptosis. Thus, nutrient restriction may encourage apoptosis of infected cells. Nutrient restriction can thereby offer protection by simultaneously limiting nutrients to both the host cells and the infectious organisms.

The nomenclature of cell death: recommendations of an ad hoc Committee of the Society of Toxicologic Pathologists.

The last several years have seen considerable confusion regarding the terms "apoptosis" and "necrosis" in pathology. This situation prompted the Society of Toxicologic Pathologists to charter the Committee on the Nomenclature of Cell Death, which was charged with making recommendations about the use of the terms "apoptosis" and "necrosis" in toxicity studies. The Committee recommends use of the term "necrosis" to describe findings comprising dead cells in histological sections, regardless of the pathway by which the cells died. The modifiers "apoptotic" and "oncotic" or "mixed apoptotic and oncotic" are recommended to specify the predominant morphological cell death pathway or pathways, when appropriate. Other standard modifiers, indicating the lesion distribution and severity, may also be used in conjunction with these. "Individual cell necrosis" (also known as "single cell necrosis") may be either of the apoptotic, oncotic, or mixed types. In many cases, more traditional terms such as "coagulation necrosis" may be used to convey a meaning similar to oncotic necrosis. It is important that pathologists use terms that accurately and concisely convey the level of information appropriate to the study's needs. Furthermore, toxicologic pathologists should actively help to disseminate these recommendations to other biologists and to regulatory authorities.

Preclinical promise of becaplermin (rhPDGF-BB) in wound healing.

Advances in molecular biology have made possible the production of highly purified recombinant human proteins, and recombinant human growth factors have emerged as potential therapeutic wound healing agents. Becaplermin (recombinant human platelet-derived growth factor-BB [rhPDGF-BB]) quickly emerged as one of the leading candidates for clinical trials. Before the expected therapeutic potential of rhPDGF-BB and other growth factors could be realized, a number of concerns had to be addressed (eg, would growth factors show effects in normal animals, what parameters of wound healing would be affected, and would quality of healed wounds be normal?). In animal models, rhPDGF-BB demonstrated wound healing activity, predominantly by enhancing the formation of granulation tissue, but it was not known whether this effect on granulation tissue would translate into enhanced healing of chronic skin ulcers in humans. The objective of this article is to review how the study of rhPDGF-BB in animal wound healing models has assisted in addressing the potential clinical utility of rhPDGF-BB. Results of animal studies are summarized, and the advantages and limitations of the animal models are discussed.

An adaptationist view of apoptosis.

A cell's decision whether to undergo apoptosis (cell suicide) is examined here from an adaptationist perspective, rather than a mechanistic one. External and internal inputs to the cell's protein-based information processing network are used in making this decision, with the cell factoring in its replaceability. A system in which each cell takes primary responsibility for deciding its own fate has great adaptive value because it harnesses each cell's self-knowledge rather than waiting for external cues to be recognized by other cells. Cell self-destruction can be an important selective mechanism, potentially leading to better performance of tissues over time. However, reliance on cells to monitor themselves has a flaw, since cells may incur selfish mutations that impair their apoptotic responsibility. The tight control exerted over somatic cells serves to check selfish genes involved in neoplasia and viral infections. Germ cells appear to be similarly monitored, both by other germ cells and by supporting follicular or Sertoli cells, thus maintaining the advantages offered by an apoptotic system. The adaptationist approach views the limited replacement of neurons and cardiac myocytes as likely to have net survival value. The linkage of these cells into a network with their neighbors throughout a lifetime allows for a precisely functioning team of cells expected to compensate for gradual declines in individual cell functionality. Replacement of apoptotic cells with naive cells might decrease brain functionality and might risk upsetting the conduction of cardiac impulses. The evolutionary viewpoint lends itself to new hypotheses, but only the boldest speculator would have predicted a system in which cells are given primary responsibility for deciding whether to kill themselves when they deem it beneficial to the organism.

Assessment of full-thickness wounds in the genetically diabetic mouse for suitability as a wound healing model.

While using the diabetic C57BL/KsJ db/db mouse as a wound healing model, we encountered several repair patterns which affect its suitability as a predictive screening model for certain indications. For example, wound contraction, albeit impaired, was found to be particularly dependent on bandaging technique and vehicle type. Wounds which had been continuously occluded with Opsite dressings had a high relative variability in contraction, and there was a tendency toward reduced contraction, suggesting that the dressings were acting as a splint. Viscous dosing vehicles inhibited contraction of occluded wounds but appeared to enhance contraction of nonoccluded wounds. In contrast to many other models, occlusion in these studies did not enhance reepithelialization when compared with air exposure (the rate of reepithelialization in db/db mice appeared normal, typically growing 2 mm from each edge in 10 days). Also in contrast to other wound healing models, viscous dosing vehicles when used under occlusion inhibited reepithelialization. However, as seen in other wound healing models, granulation tissue thickness was reliably increased in response to treatment with recombinant human platelet-derived growth factor-BB. Our experience with the db/db diabetic mouse model has led us to recommend the use of this animal model only after its limitations have been identified and accepted.

Evaluation of ibuprofen for pressure ulcer prevention: application of a rat pressure ulcer model.

This blinded study describes the effect of ibuprofen (IBU) on experimental pressure ulcers (PU) induced in the fuzzy rat model. Ibuprofen's fibrinolytic activity has been found effective in preserving dermal vasculature in rats following burn injury. Experimental pressure ulcers were generated on the hips of fuzzy rats with the aid of computer control, using five daily, 6-hour pressure sessions. In the first study, 17 rats received intraperitoneal (I.P.) injection of IBU or saline control after each pressure session. In the second study, 44 rats received IBU or control, before, during, and after application of pressure by I.P. or intramuscular (I.M.) injections. Quantitation of histology is based on five prominent lesions, including: ulceration, infarction, panniculus carnosus necrosis, fat atrophy, and deep muscle necrosis. The following hypothesis was tested: Reperfusion injury and vascular occlusion in pressure ulcers may be similar to that which occurs in burns and, therefore, is responsive to IBU intervention. The results indicate that IBU does not have significant effects on the development of pressure ulcers. The mechanisms of IBU action in other organ and tissue systems are discussed in relation to the effect of IBU on the pressure ulcer model.

Comparative efficacy of nonsteroidal anti-inflammatory drugs and anti-thromboxane agents in a rabbit adhesion-prevention model.

A variety of nonsteroidal anti-inflammatory drugs (NSAIDs) has been found to inhibit postsurgical peritoneal adhesion formation in a number of animal models. A rabbit uterine horn adhesion model was used to directly compare several commonly used NSAIDs of different chemical classes in a single animal study to evaluate their ability to prevent adhesion formation. The effect of thromboxane inhibitors on adhesion prevention was also evaluated. Each of the NSAIDs tested (tolmetin, ibuprofen, aspirin, and indomethacin) showed significant and comparable efficacy. In this same study, imidazole, a thromboxane synthetase inhibitor, also showed significant efficacy. In a second study, ridogrel, an inhibitor of thromboxane synthetase as well as a thromboxane A2 receptor blocker, also showed significant efficacy in reducing peritoneal adhesion severity. These results further support the view that NSAIDs act to prevent adhesions through a common mechanism. In addition, thromboxane A2 inhibitors were also shown to be efficacious in adhesion prevention, suggesting that platelets may play a substantial role in adhesion formation.

An animal model and computer-controlled surface pressure delivery system for the production of pressure ulcers.

Pressure ulcers continue to be a major health care problem. This paper describes an animal model and surface pressure delivery system for the production of experimentally derived pressure ulcers. A method for inducing dermal pressure lesions on the fuzzy rat was developed using a computer-controlled displacement column which produced a constant tissue interface pressure. The pressure column consists of a force transducer located between two 0.5-in (1.27-cm) diameter metal cylinders. The desired cutaneous pressure is maintained by a computer-controlled miniature stepper motor which displaces the column with the aid of interactive software. The force transducer signal is converted from analog to digital form, amplified, and recorded. Blood perfusion is monitored using a laser Doppler flowmeter (located in the tip of the column) during the application of pressure. The application of 145 mmHg pressure for 5 consecutive 6-hr sessions resulted in a greater than 90% incidence of pressure ulcers. The implications of our model and contributions of earlier animal models are discussed. This model provides a tightly controlled and measured environment making possible the scientific study of ulcer development and the evaluation of potential preventative or curative compounds.

Histopathology of pressure ulcers as a result of sequential computer-controlled pressure sessions in a fuzzy rat model.

This study describes the sequential histopathological changes that occur in the development of pressure ulcers experimentally induced in the fuzzy rat model. Computer-controlled pressure was applied for six hours at a maximum of five sessions, to skin over the greater trochanter of anesthetized rats. Lesions were similar, but more pronounced after the third, fourth, and fifth sessions as compared to the first or second sessions. Lesions developed first in the muscle rather than the dermis or epidermis. The lesion most often associated with pressure was necrosis of the panniculus carnosus muscle, often accompanied by damage to underlying adipose tissue. Recurrent pressure results in increasingly severe damage to the vascular system and parenchyma, consistent with an ischemia/reperfusion insult initiated through a free radical mechanism.

Efficacy of tolmetin sodium for adhesion prevention in rabbit and rat models.

Tolmetin sodium's efficacy in preventing primary adhesions was evaluated in two adhesion models each in two species: (1) rabbit uterine horn, (2) rat uterine horn, (3) rabbit peritoneal side wall, and (4) rat peritoneal side wall. In each model a single instillation of tolmetin sodium solution into the peritoneal cavity at the time of surgery effectively reduced adhesion formation. This efficacy extended over a wide range of concentrations, volumes, and total dosages, and was similar in rabbits and rats. An aqueous solution of 1 mg/ml tolmetin sodium in 5-15 ml in rabbits and in 3 ml in rats was consistently efficacious in reducing postoperative adhesion formation.

Dose responsive effects of PDGF-BB, PDGF-AA, EGF, and bFGF on granulation tissue in a guinea pig partial thickness skin excision model.

A guinea pig partial thickness skin excision model was used to evaluate the effects of recombinant human PDGF-BB, PDGF-AA, EGF, and bFGF on granulation tissue (neodermis) formation. These growth factors tended to increase the thickness of the granulation tissue bed when assessed histologically at day 7. Using only four animals per group, PDGF-BB at 30 and 100 micrograms/ml consistently and significantly increased the thickness of the granulation bed 2-3 times that of control. Except for the increased thickness, the granulation tissue appeared normal. PDGF-AA and EGF also significantly increased the granulation tissue thickness, and bFGF gave indications of an effect. There was no evidence of synergistic effects between PDGF-BB, EGF, and/or bFGF.

Evaluation of PDGF-BB, PDGF-AA, bFGF, IL-1, and EGF dose responses in polyvinyl alcohol sponge implants assessed by a rapid histologic method.

Polyvinyl alcohol sponge implants were used in rats, mice, and guinea pigs to determine dose responses of growth factors. Eight differently treated sponges per rat or guinea pig (4/mouse) were injected with test material on alternate days and evaluated at day 8. Much of the observed response occurred in and around the capsule and was manifest as densely cellular granulation tissue. Including this capsular response in a single histologic slide ranking system provided a more sensitive and faster method of assessing growth factor effects than measurement of connective tissue ingrowth alone. Clear dose responsive effects were seen with recombinant human PDGF-BB, PDGF-AA, bFGF, and IL-1 beta, while EGF gave a lesser response. Lipopolysaccharide did not affect the connective tissue response, alone or in combination with PDGF-BB. PDGF-BB was tested in each species, and the dose response characteristics were qualitatively and quantitatively similar across species.

Dilatation in anastomosed arteries can be an artifact of explantation.

Fusiform dilation of the anastomotic site was found at explantation in the majority of rabbit femoral arteries anastomosed with newly developed microclips. This observation was also noted at sutured anastomoses. The mechanism responsible for this morphologic finding was studied by the following experiments: 1) measuring and explanting anastomosed arteries relaxed with 20% lidocaine, 2) latex casts of microclipped arteries, 3) comparing angiographic and histologic morphometric data, and 4) examining synthetic vascular graft anastomoses with identical dilatation. The observations demonstrate that trauma associated with explantation (direct dissection and fixative perfusion) leads to arterial vasoconstriction, but focal medial damage at the anastomotic site prevents constriction at this site.

An evolutionary perspective of endotoxin: a signal for a well-adapted defense system.

In the evolutionary view of endotoxin presented here, endotoxin is the primary signal animals use to detect gram negative (Gr-) bacteria. Since endotoxin, or lipopolysaccharide (LPS), is an integral part of the surface of all Gr- bacteria, it was excellent evolutionary 'choice' for the signal. The concept of an 'endotoxin response system' (ERS) is introduced. The ERS protects against Gr- bacteria by employing many of the body's defenses to both detect and react against LPS. The intensity of the response has evolved to maximize protection while minimizing the biological cost and self-damaging effects. The setting of the response, here termed the 'endostat', is programmed by natural selection and fine tuned by feedback mechanisms. Other potentially invasive organisms are detected by different signals, but the effector components of the defenses are similar. This evolutionary view of LPS offers a framework for the seemingly contradictory findings on endotoxin and suggests new avenues of productive research.