Chronic Diseases as Barriers to Oxygen Delivery: A Unifying Hypothesis of Tissue Reoxygenation Therapy.

Modern medical practice has resulted in the accumulation of a growing number of incurable chronic diseases, many of which are inflammatory in nature. Inflammation establishes a hypoxic microenvironment within tissues, a condition of inflammatory hypoxia (IH). Tissues thus affected become severely compromised, are unable to elicit adaptive responses and eventually develop fibrosis and fixed microvascular deficits. Previous work has demonstrated that tissue hypoxia exits even within the simple human model of self-resolving inflammation, the tuberculin reaction. Failed resolution of IH establishes a vicious cycle within tissues that perpetuates tissue hypoxia and resists standard drug therapies. Diseases such as sepsis, chronic cutaneous wounds, kidney disease, traumatic brain injury, solid tumors, inflammatory bowel disease, and chronic bacterial infections (urinary tract infection, cystic fibrosis) are tissue specific manifestations of chronic IH. Successful reversal of IH, through tissue re-oxygenation therapy (TROT), will break this vicious cycle and restore tissue homeostasis. The examples of solid tumors and inflammatory bowel disease are presented to illustrate a theoretical framework to support this hypothesis. Re-oxygenation of compromised tissues must occur before successful treatment of these diverse chronic disease s can be expected.

Preoperative Stress Conditioning in Humans: Is Oxygen the Drug of Choice?

Complications following invasive medical and surgical procedures are common and costly. No clinical protocols exist to actively condition patients prior to these high risk interventions. Effective preconditioning algorithms have been repeatedly demonstrated in animal models for more than a quarter century, where brief exposures to hyperthermia (heat shock), ischemia (ischemic preconditioning) or hypoxia have been employed. Heat shock pretreatment confers protection against experimental acute ischemia-reperfusion, endotoxin challenge and other stressors. The resulting state of protection is short lived (hours) and is associated with new gene expression, typical of a cell stress response (CSR). We aim to use the CSR to actively precondition patients before surgery, a process termed stress conditioning (SC). SC is a procedure in which tissues are briefly exposed to a conditioning stressor and recovered to permit the development of a transient state of resistance to ischemia-reperfusion injury. Successful SC of humans prior to surgery may reduce postoperative complications related to periods of hypotension, hypoxia, or ischemia. Stressors such as heat shock, acute ischemia, endotoxin, heavy metals or hypoxia can induce this protected state but are themselves harmful and of limited clinical utility. The identification of a stressor that could induce the CSR in a non-harmful manner seemed unlikely, until high dose oxygen was considered. Human microvascular endothelial cells (HMEC-1) exposed to high dose oxygen at 2.4 ATA × 60-90 min developed increased resistance to an oxidant challenge in vitro (peroxide). The molecular changes described here, together with our understanding of the CSR and SC phenomena, suggest high dose oxygen may be the drug of choice for clinical preconditioning protocols and should be systematically tested in clinical trials. Oxygen dosing includes the following ranges: room air exposure is 0.21 ATA, clinical oxygen therapy 0.3-1.0 ATA (normobaric hyperoxia) and hyperbaric oxygen is 1.5-3.0 ATA (ATA-atmosphere absolute).

Animals bearing malignant grafts reject normal grafts that express through gene transfer the same antigen.

Breaking the state of immunological unresponsiveness of tumor-bearing individuals to cancer is a prerequisite for active or passive tumor-specific immunotherapy. To study this problem the immunogenic MHC class I antigen, K216 was transfected into a progressor tumor. The transfected tumors were regularly rejected by normal mice but grew progressively in mice bearing nontransfected tumors. In addition, transgenic mice were derived to obtain normal cells and tissues expressing the same K216 gene product. Normal mice rejected K216-positive normal or malignant tissue grafts and generated K216-specific CTL in vitro and in vivo in response to these challenges. In contrast, mice bearing nontransfected tumors, though rejecting K216-positive nonmalignant tissue grafts, did not reject K216-positive tumors nor generate K216-specific CTL in response to K216-positive tumor cells. Mice bearing K216-positive tumors also rejected the nonmalignant K216-positive tissue grafts, but this in vivo response failed to lead to rejection of the simultaneously present tumor graft expressing the same antigen; in fact, immunity had no measurable effect whatsoever on tumor size or incidence and caused no selection for antigen loss variants. Taken together, the present findings suggest that transfer of expression of a target antigen into nonmalignant cells provides a way for obtaining effective stimulation of antigen-specific CTL in tumor-bearing mice, but that additional manipulations will be required to cause immunological rejection of established tumors.

Babesiosis in a renal transplant recipient acquired through blood transfusion.

BACKGROUND: The success of organ-replacement therapies has resulted in a population of chronically immunosuppressed but active people who experience increased vulnerability to tick-borne zoonoses. Several of these infections may be life threatening. Human babesiosis is an emerging zoonosis that is transmitted by the same tick that transmits Lyme disease and human granulocytic ehrlichiosis. METHODS: We briefly review these zoonoses and present a case of a renal transplant recipient who survived infection by Babesia microti contracted through blood transfusion. RESULTS: A recipient of a living-related renal transplant developed acute postoperative hemolytic anemia. The etiology of this anemia was diagnosed by peripheral red blood cell smear as Babesia microti. The patient was managed by a reduction in transplant immunosuppressive therapy and administration of clindamycin and quinine antimicrobials. CONCLUSIONS: Transplant patients may contract babesiosis after tick exposure and/or via blood transfusion. The diagnosis of babesiosis may be confused with malaria and should be included in the differential diagnosis of posttransplant hemolytic-uremic syndrome in organ transplant patients.

Tissue-level cytoprotection.

In vitro and ex vivo tissue models provide a useful level of biological organization for cytoprotection studies positioned between cultured cells and intact animals. We have used 2 such models, primary tissue cultures of winter flounder renal secretory epithelium and ex vivo preparations of rat intestinal tissues, the latter to access the microcirculation of exposed mesentery tissues. Herein we discuss studies indicating that differentiated functions are altered in thermotolerant or cytoprotected tissues. These functions include transepithelial transport in renal epithelium and attachment and transmigration of leukocytes across vascular endothelium in response to mediators of inflammation. Evidence pointing to inflammation as a major venue for the heat shock response in vertebrates continues to mount. One such venue is wound healing. Heat shock proteins are induced early in wound responses, and some are released into the extracellular wound fluid where they appear to function as proinflammatory cytokines. However, within responding cells in the wound, heat shock proteins contribute to the acquisition of a state of cytoprotection that protects cells from the hostile environment of the wound, an environment created to destroy pathogens and essentially sterilize the wound. We propose that the cytoprotected state is an anti-inflammatory state that contributes to limiting the inflammatory response; that is, it serves as a brake on inflammation.

Effects of heat shock, stannous chloride, and gallium nitrate on the rat inflammatory response.

Heat and a variety of other stressors cause mammalian cells and tissues to acquire cytoprotection. This transient state of altered cellular physiology is nonproliferative and antiapoptotic. In this study, male Wistar rats were stress conditioned with either stannous chloride or gallium nitrate, which have immunosuppressive effects in vivo and in vitro, or heat shock, the most intensively studied inducer of cytoprotection. The early stages of inflammation in response to topical suffusion of mesentery tissue with formyl-methionyl-leucyl-phenylalanine (FMLP) were monitored using intravital microscopy. Microvascular hemodynamics (venular diameter, red blood cell velocity [Vrbc], white blood cell [WBC] flux, and leukocyte-endothelial adhesion [LEA]) were used as indicators of inflammation, and tissue levels of inducible Hsp70, determined using immunoblot assays, provided a marker of cytoprotection. None of the experimental treatments blocked decreases in WBC flux during FMLP suffusion, an indicator of increased low-affinity interactions between leukocytes and vascular endothelium known as rolling adhesion. During FMLP suffusion LEA, an indicator of firm attachment between leukocytes and vascular endothelial cells increased in placebo and gallium nitrate-treated animals but not in heat- and stannous chloride-treated animals, an anti-inflammatory effect. Hsp70 was not detected in aortic tissue from placebo and gallium nitrate-treated animals, indicating that Hsp70-dependent cytoprotection was not present. In contrast, Hsp70 was detected in aortic tissues from heat- and stannous chloride-treated animals, indicating that these tissues were in a cytoprotected state that was also an anti-inflammatory state.

Surgical stress and the heat shock response: in vivo models of stress conditioning.

All forms of surgical therapy are stressful and injurious. The majority of surgical procedures are performed electively and provide an opportunity to condition the patient before surgery to maximize outcome. We have successfully protected the spinal cord and kidneys from warm ischemia-reperfusion injury with whole-body heat shock (42.5 degrees C, 15 min, HS) and recovery (37 degrees C, 6-8 h) before acute aortic occlusion. Control rabbits experienced an 88% incidence of paralysis (7/8) after acute spinal cord ischemia, while HS-pretreated animals never became paralyzed (0/9, p < 0.001). Control pig kidneys showed partial function (4/8 survival) after 90-min warm ischemia, while HS-pretreated kidneys always functioned (8/8 survival, p < 0.04). A positive temporal association was made between the HS-associated functional protection and the enhanced expression of inducible HSP70. The induction of the heat-shock response (cellular stress response) to protect tissues from lethal acute ischemia-reperfusion injury could be employed in a wide range of medical and surgical settings.

Intrahepatic portosystemic vascular stents: a bridge to hepatic transplantation.

Refractory esophageal variceal hemorrhage (EVH) remains a formidable problem in patients awaiting liver transplantations. Transjugular intrahepatic portosystemic shunts (TIPS) have provided an alternative approach for managing EVH that may obviate the need for portosystemic shunt surgery. Experience with TIPS placement and subsequent successful hepatic transplantation in patients without previous portosystemic shunt surgery has not been previously reported. Two patients are reported who underwent TIPS placement and subsequent successful hepatic transplantation without previous portosystemic shunt surgery. This experience indicates that (1) TIPS can provide effective control of EVH for at least several weeks, (2) TIPS placement decreases portal hypertension, thus facilitating technical performance of the transplant procedure and minimizing blood loss, (3) TIPS may undergo vascular incorporation, thus requiring that they be accurately positioned so that the lengths of suprahepatic inferior vena cava and portal vein are not compromised at the time of transplantation, (4) TIPS thrombosis can be effectively treated and prolonged patency may be observed, and (5) deterioration in hepatic function and exacerbation of hepatic encephalopathy were not observed after TIPS placement. In summary, TIPS provide an attractive, effective means for managing refractory EVH in patients awaiting liver transplantation.

Acute stress impairs lymphocyte function but spares pancreatic endocrine function.

In the same animal, following HS and recovery, pancreatic islet function remains intact while immunologic functions are impaired. Cellular responses to thermal stresses are complex and tissue specific.

Albumin improves islet isolation: specific versus nonspecific effects.

1. BSA-containing solutions improve islet yields using standard collagenase digestion techniques. 2. The BSA effect on islet isolation is independent of source and lot of collagenase. 3. The BSA effect on islet isolation is not due solely to its colloid action, as HES failed to achieve the same level of improvement seen with albumin. 4. BSA can protect islets from warm ischemic injury, and the protective action appears to be unique to albumin, as HES was not as effective.

Improving acute skin-flap survival through stress conditioning using heat shock and recovery.

We present our initial experience with a new method of increasing the survival of acute skin flaps through stress conditioning using heat shock and recovery. The heat-shock response is a basic form of stress response that exists on the cellular level. When cultured cells or whole organisms are exposed to supraphysiologic levels of heat, they respond by synthesizing a number of highly conserved proteins known as heat-shock proteins. These proteins have been shown to offer the cell or organism a survival advantage over nonstressed controls. The study demonstrates a significant survival advantage in acute dorsal skin flaps of Sprague-Dawley rats (p = 0.001). Study animals (n = 10) were subjected to a heating blanket set at 45 degrees C for 30 minutes and were allowed 6 hours' recovery before developing the flaps. Heat-shock protein was demonstrated in immunohistochemically stained sections of skin from the study animals but not in control animal skin (n = 14). We postulate that through stress conditioning a latent mechanism present within all cells was activated, thereby allowing the cells of our experimental flaps to better survive the stress of the acute flap model.

Stress conditioning: a novel approach to organ preservation.

Effective stress conditioning can extend the safe cold storage time of rat kidneys to 48 hours. We demonstrated that planned induction of the stress response, by heat shock, can be used to stress condition the transplant organ and protect it against the damages of cold storage. A powerful and useful protective mechanism exists in a latent form in all cells. This mechanism can be rapidly activated in a controlled and planned way to allow tissues temporarily to resist injury. Through stress conditioning it is possible to provide a high level of protection in situations in which stress can be anticipated and planned for.

Organ transplantation at the Hartford Transplant Center.

Over 1,243 organ transplants have been performed at the Hartford Transplant Center over the past two decades. Survival in kidney, heart, liver, and pancreas patients is at or above the national average. Hartford was one of the first centers to use triple immunosuppression, which significantly improved survival in kidney transplantation. For recipients of kidneys from living related donors and cadaveric kidneys, two-year actuarial graft survival has been 98% and 83%, respectively, over the last five years. For heart and liver transplants, two-year survival has been 79% and 67%, respectively. Despite high success rates at most transplant centers, donor organs remain scarce. This problem needs to be addressed through increased cooperative efforts in the health-care community and the general public.