Abstract

We have followed-up with health monitoring from 1976 to 1998 thousands of people affected by the fallout of TCDD over Seveso and the nearby area which occurred on July 10, 1976. These data present advantages in that: (1) they were derived from individuals of both sexes covering all age ranges; (2) since then serum samples have been kept frozen and we have been recently able to measure (at CDC, Atlanta, USA) the TCDD blood lipid content. We can now therefore directly correlate exposure with health effects during the years. The results indicate that:
-serum TCDD levels in residents showed a very elevated exposure (up to 56,000 ppt);
-chloracne was the only clinical alternation positively correlated to TCDD contamination levels, even if not completely, and with differing individual susceptibility;
-miscarriages, perinatal mortality, low birthweight or congenital malformations did not significantly increase;
-clinical monitoring of children and adults did not prove any clear association between morbidity (except chloracne) and TCDD exposure;
-laboratory results showed minimal differences between exposed (even if hugely exposed) and controls in the period of acute exposure (1976-1977) with relation to liver function tests, complement haemolytic activity, white blood cells, lymphocytes and haemoglobin. These differences were subclinical, faced and then disappeared with time.
A slight increase of leukemias, soft tissue sarcomas and rectal cancer in males has been observed after 20 years. Part of the exposed people were controlled in 1992-1998 and the results showed that:
-no laboratory pathology was related to TCDD levels in both the acute and chronic phase;
-cytochrome P450 IA2 did seem to be induced after about 17 years in exposed people compared to controls as measured by the Caffein Breath Test;
-the half-life of TCDD was longer in women (about 9 years) than in men (about 7.5 years), while in children it is much shorter.
A striking skewing of sex ratio at birth (males/males + females) with excess of female (p < 0.001) from parents highly exposed to TCDD has been described for the period 1977-84. Very recently this effect has been shown to be permanently related only to father exposure with the pre and puberty period being a very sensitive period and it has been recently confirmed in a dioxin induced chloracneic Austrian cohort. The TCDD concentrations by which this lower sex ratio is induced in Seveso group are only about 20 times the estimated average concentration corrently found in human beings in industrialized countries. The human male reproductive system is demonstrated for the first time to be very sensitive to dioxin. This fact can have important public-health implications due to the very different individual sensitivity in humans. (Presented at the 1197th Meeting, February 5, 2001.)

Tissue engineering is an emerging technology to regenerate or reconstruct biological tissues or organs from cells. Therefore, engineered tissues are completely different from artificial organs made from man-made materials, but rather similar to tissues and organs used for current organ transplantation if allogeneic cells are employed for the tissue engineering. However, engineered tissues are autogeneic and do not require any immunosuppressive agents if patient's own cells are used for the tissue regeneration or reconstruction. If diseased or lost tissues are too large in size to regenerate from simple cell transplantation, we should provide artificial extracellular matrix to the place where the tissue regeneration is under way, because of the loss of natural extracellular matrix through injury or disease. In most cases, tissue engineering requires artificial extracellular matrix in addition to cells. Generally, the artificial extracellular matrix is called scaffold and consists of three-dimensional, porous, and absorbable materials. When the tissue regeneration is accomplished as expected, the provided scaffold is no more necessary or rather disturbs the tissue regeneration, if it remains there without resorption. This is a major reason why absorbable materials are necessary for tissue engineering. There are natural and synthetic, absorbable biomaterials. The representative materials include collagen and fibrin as natural and polyglycolide (PGA) and polylactide (PLA) as synthetic biomaterials. Based on their physical and biological characters, we select the most suitable one from a variety of absorbable biomaterials for the tissue engineering we aim at. This presentation demonstrates how absorbable materisls are important in tissue engineering as scaffold for cells and, in addition, carrier for sustained release of cell growth factors. (Presented at the 1218th Meeting, July 11, 2001.)

In small intestinal crypts, α-defensins, termed cryptdins, are abundant constituents of Paneth cell secretory granules, and the peptides are highly microbicidal when assayed in vitro. Cryptdin precursors are processed and activated in mouse Paneth cells by MMP-7, and MMP-7 null mice lack mature cryptdins and have a measurable defect in clearing oral bacterial infections and are more susceptible to lethal systemic disease caused by Salmonella typhimurium. Mouse and human Paneth cells secrete microbicidal peptides when exposed to bacteria or bacterial antigens. In mice, the dose-dependent secretion occurs within minutes, and cryptdins account for 70% of the released bactericidal peptide activity. Gram negative or Gram positive bacteria, lipopolysaccharide, lipoteichoic acid, lipid A, and muramyl dipeptide elicit cryptdin secretion, but live fungi and protozoa do not. Thus, intestinal Paneth cells contribute to innate immunity by sensing bacteria and bacterial antigens via receptor-mediated mechanisms and discharging microbicidal peptides at effective concentrations. The intermediate conductance Ca⁺⁺-activated K⁺ channel, mIKCa1, modulates Paneth cell secretory responses to bacteria and pharmacologic stimulation. RT-PCR studies performed on isolated crypts and individual Paneth cells showed that the mIKCa1 gene is expressed by Paneth cells in mouse small bowel, and IKCa channel antagonists clotrimazole, charybdotoxin, and highly selective triarylmethane inhibitors blocked Paneth cell secretion induced by bacteria, lipopolysaccharide (LPS), or carbamyl choline by approximately 50%. These findings show that the Ca⁺⁺-gated mIKCa1 channel must function to allow Paneth cell secretion to occur at maximal levels. Thus, by modulating K⁺ efflux in response to the influx of extracellular Ca⁺⁺ mIKCa1 regulates the magnitude of Paneth cell secretion with effects on enteric innate immunity. (Presented at the 1219th Meeting, July 10, 2001.)

Traditionally, the various categories of psychoactive drug have been regarded as quite distinct from each other with respect to pharmacological mechanisms of action, as shown by lack of generalization of discriminative stimulus properties, lack of cross-tolerance, and difference of dependence liability and optimum approaches to treatment. However, recent research has revealed a surprising number of mechanisms common to all these agents. They all activate the same mesolimbic dopaminergic "reinforcement" pathway, and various neurotransmitters affect their reinforcing properties in the same ways. Selective serotonin reuptake inhibitors, and ondansetron and other 5-HT₃ antagonists, decrease the oral and intravenous intake of morphine, ethanol and amphetamine, and also decrease drug-conditioned place preference (CPP) and other signs of reinforcement. Similarly, morphine increases ethanol intake, whereas the mu receptor blocker naltrexone and the delta blocker naltrindole act centrally to decrease drug-induced release of dopamine in the N. accumbens as well as oral or parenteral selfadministration of ethanol, opiates and nicotine. The endogenous anti-opioid peptide nociceptin decreases both ethanol consumption and morphine-induced CPP. Ethanol, morphine and various other drugs have similar effects on intracellular second messenger and effector systems, such as type 7 adenylyl cyclase, cell membrane Na,K-ATPase, calcium L channels, protein kinases and nuclear transcription factors. Indeed, several groups have found that cross-tolerance between ethanol and morphine does occur on tests which are affected similarly by both drugs, and the NMDA-receptor blocker MK-801 inhibits the development of tolerance to high doses of both ethanol and morphine, as well as sensitization by low doses of each or by cocaine. Acute doses of ethanol, diazepam, morphine or nicotine have no effect on the brain levels of diazepam binding inhibitor (DBI), but all of them, when administered chronically, raise the levels of DBI and of its mRNA similarly, especially during their respective withdrawal reactions. Thus, although these drugs do differ in their spectra of acute effects and withdrawal patterns as a function of differences in their respective receptor distributions, they share many post-receptor effects that are basic to their reinforcing effects, tolerance and dependence. These shared actions and effects suggest that newer pharmacotherapies of dependence may become closely similar for all these drugs. (Presented at the 1224th Meeting, August 27, 2001.)

Blood substitutes were originally formulated to simulate blood's oxygen carrying capacity, viscosity, p50, and colloid osmotic pressure, under the hypothesis that blood is the most desirable fluid in volume restitution. However, hemorrhage affects microvascular function causing decreased functional capillary density, and lowered tissue oxygenation, conditions not universally reversed with blood retransfusion. Restoration of microvascular function is seldom complete upon retransfusion of blood. New formulations of hemoglobin molecules in solutions whose oncotic pressure is 60-100 mmHg, p50 is 5 mmHg, viscosity 3-4 cP, and oxygen carrying capacity is 4-7 g/dl equivalent hemoglobin produce better microvascular function after resuscitation relative to whole blood and fluids with transport properties similar to blood. This is due to increased plasma viscosity which increases capillary transmural pressure, reversing the capillary collapse induced during low perfusion pressure, and high oncotic pressure which reinforces this effect, since it brings more fluid into the circulation. Microvascular studies of hemorrhagic shock resuscitation show that functional capillary density is a primary determinant of survival, as critical as insuring oxygen supply, since closed capillaries lead to the accumulation of byproducts of metabolism which ultimately become toxic. The needed properties are achieved by conjugating hemoglobin and polyethylene glycol. Resuscitation fluids based on hemoglobin containing vesicles may provide the next level of functional improvement in the formulation of volume restitution fluids since their biophysical properties can be specifically controlled. (Presented at the 1226th Meeting, September 6, 2001.)

In this talk I will try to address the following question: why is it that during a scienti.c or medical lecture most of the audience is either asleep or thinking about sex, or both? My hypothesis is that this unfortunate (for the speaker) state of affairs is because so many lectures are incomprehensible, boring or both, oftentimes because little thought has been given to what makes a presentation good or bad. In other words, many speakers concentrate on their data but not on their presentation. I will cover some of the common lecture mistakes such as excessive ego, not thinking about the audience, not being clear on what you want to convey, poor slides, ineffective use of slides, and other miscellaneous, but common, problems with presentations. I will give my opinion about controversial subjects such as dual projection, slide backgrounds, and whether it is appropriate to show a picture of your dog during your talk. Finally, I will conclude with the observation that a talk is not a paper and must be handled differently in many respects. I expect this talk to be controversial and to generate discussion. (Presented at the 1227th Meeting, September 11, 2001.)

The factors that account for differences in asthma severity have been the subject of intense investigation. Our investigations have focused on understanding the biological actions of asthma mediators, quantifying their expression in the disease, and determining their ability to account for differences in asthma severity. Shortly after the protein sequence of guinea pig eotaxin was reported, we cloned guinea pig eotaxin and demonstrated that after allergen sensitization and challenge its expression is increased in airway epithelial cells and endothelial cells in the lung. To extend these findings to the human system, we examined the ability of asthmarelated cytokines to induce eotaxin expression in cultured human airway epithelial cells and cell lines. We found that airway epithelial cells responded to primary cytokines with a rapid and transient increase in eotaxin mRNA and protein expression, with a pattern that was similar to that observed in the guinea pig and mouse models of allergic airway inflmmation. We next validated our animal experiments by demonstrating that segmental allergen challenge in allergic asthmatics is associated with a signifiant increase in eotaxin expression that correlated with eosinophil recruitment 4 hours after allergen challenge. In accomplishing this study, we became aware that eotaxin expression is systemically detectable and determined that plasma levels of eotaxin had predictive power for asthma diagnosis and severity as judged by baseline FEV₁. The predictive power of eotaxin was independent of the degree of sensitization as judged by IgE level or the ability to mobilize peripheral inflmatory cells as judged by eosinophil counts. While these findings associated eotaxin with asthma severity, they could not distinguish whether eotaxin was directly harming the lung or if it was mobilized by lungs that were damaged by other mediators. To make this distinction we examined the role of a genetic variant that limits the systemic expression and cellular secretion of eotaxin. We found that individuals with the expression-limiting form of the eotaxin gene had better lung function than those with the robust eotaxin-expressing form. The findings of this study suggest that eotaxin is important for the longitudinal loss of lung function in the asthmatic lung. (Presented at the 1228th Meeting, September 20, 2001.)

Clinical trials of novel anti-sepsis agents have proven to be a difficult and daunting challenge. The complex interactions between underlying disease processes, infecting microbial pathogen(s), and host innate immune response generates intrinsic heterogeneity among patient populations with severe sepsis. This has contributed to the inconsistent and often disappointing results in many, large, multi-center clinical trials in sepsis. Despite numerous setbacks, major advances are being made in the understanding of the molecular basis of systemic inflammation and coagulopathy in sepsis over the past two decades. Several recent phase III sepsis trials have met with some success including: the French low-dose steroid trial; the anti-TNF Fab fragment monoclonal antibody study; and, most notably the PROWESS trial with recombinant human activated protein C. Several other phase II trials have shown promise including the platelet activating factor-acetyl hydrolase study, the soluble phospholipase A₂ trial, and the tissue factor pathway inhibitor trial. Several of these agents are now in advanced stage, clinical trials in sepsis patients. Other approaches are in various stages of clinical investigation such as anti-endotoxin strategies (Lipid A antagonists, CD14 monoclonal antibody, phospholipid binding agents, endotoxin binding columns, anti-endotoxin vaccines); anti-cytokines and host derived mediator inhibitors (small molecule cytokine inhibitors, anti-inflammatory cytokines, neutrophil elastase inhibitors, TLR inhibitors); and coagulation inhibitors (factor Xa inhibitors, antithrombin, tissue factor antibodies and inhibitors) along with many other strategies. This remains an active area of clinical research since the results of the rhAPC trial leave considerable room for improvement in the overall mortality rate attributable to sepsis. Combination therapy will utilize with multiple agents that target different pathways in the pathogenesis of sepsis. The future of sepsis research will focus upon preventative strategies and use of improved diagnostics and genomics to define risk factors and optimize treatment approaches. Targeted therapies for individual patient needs will be utilized based upon specific types of infection, alterations in host immune response and coagulation capacity and underlying disease processes. (Presented at the 1230th Meeting, September 28, 2001.)

<Introduction> Degenerative disc disease of the lumbar spine is a common disorder, and it is a major cause for low-back-bain. Research in biomechanics and biology of lumbar disc disease has contributed to better understanding of its pathogenesis and treatment. <Pathogenesis of lumbar disc disease> Although the exact pathogenesis is unknown, degenerative disc disease is mechanically induced and biologically mediated. Aging also plays a role, but lumbar disc degeneration is more commonly found in the higher axially loaded lower lumbar spine. Repetitive loading, particularly torsion, also is an important biomechanical parameter. Likewise, more advanced degenerated discs are less stiff in torsion and flexionextension as compared to normal discs. Biologically, disc cells are active in producing matrices such as proteoglycans (PG) and collagens. The intervertebral disc consists of outer annulus fibrosus (AF), which is rich in collagens that accounts for its tensile strength, and inner nucleus pulposus (NP), which contains large proteoglycans and water for resisting compression loading. The chondrocyte-like cells in AF and NP produce matrices under the control of a variety of substances including growth factors and bone morphogenetic proteins (BMP), including BMP-7 (OP-1). Catabolic process or breakdown of the matrix is mediated by various matris metalloproteinases (MMPs) and cytokines. There is balance between the anabolic and catabolic processes in the normal disc, but this balance is lost in degenerating discs. The intervertebral disc also requires nutrition, mainly by diffusion from the vertebral bodies and endplates. Therefore, trauma, cigarette smoking, and other factors that affect the endplates may affect diffusion and affect nutrition to the disc cells. It should be emphasized that pain is not necessarily correlated with morphologic or biomechanical changes in the disc, and further studies are needed to elucidate the mechanism of pain in degenerative disc disease. <Treatment of lumbar disc disease> Fortunately, most people with low-back-pain get better or respond to conservative treatment such as anti-inflammatory medications, exercises and physical therapy. For patients with severe unrelenting pain for greater than 6months, surgery may become an option. Recently intradiscal electrothermal therapy (IDET) has been introduced in the United States, and preliminary results are promising, but longer outcome data and basic science studies are needed. Surgical options include a variety of fusion techniques and more recently disc prostheses. Patient selection is the key to successful outcome. Fusion techniques include posterolateral fusion with or without pedicle screw instrumentation, posterior lumbar interbody fusion (PLIF), anterior lumbar interbody fusion (ALIF), cages, minimally invasive fusion techniques. PLIF or ALIF alone with bone grafts or cages is not biomechanically stable and give a high pseudarthrosis rate except in single level cases with a collapse disc. With the advance of BMPs, the need for rigid spinal instrumentation for degenerative disc disease may decrease in the future. The use of disc prosthesis allows motion and theoretically prevents adjacent segment degeneration, but prospective randomized controlled studies are needed prior to wide application. <Future of degenerative disc disease> More research is needed in the areas of epidemiology, ergonomics, biomechanics, imaging, and biology to improve diagnosis and treatment as wells as prevention of lumbar disc disease. Biological repair or regeneration is feasible by injection a growth factor or BMPs such as OP-1 or transplanting cells that are transfected with therapeutic DNAs by viral or nonviral gene therapy. Molecular biology also will help identify candidate genes responsible for disc degeneration and repair, and tissue engineering of the intervertebral disc may help treat lumbar disc disease biologically rather than surgery. (Presented at the 1232nd Meeting, October 15, 2001.)

CD26 is a 110 kDa surface glycoprotein with dipeptidyl peptidase IV (DPPIV) activity in its extracellular domain and is expressed on numerous tissues, including T lymphocytes. Our previous work demonstrated that CD26 has a key role in normal human T cell physiology through its involvement in key signal transduction processes, as well as its physical and functional association with critical T cell molecules, including adenosine deaminase (ADA). Our recent work now suggests that CD26 is a potential target for novel treatment modalities for T cell hematological malignancies. We demonstrated that the anti-CD26 monoclonal antibody 1F7 has in vitro and in vivo antitumor effect on human CD30+ anaplastic large cell T cell lymphoma Karpas 299, as its binding induces cell cycle arrest at G₁ associated with p21 expression. We also showed that stable Jurkat transfectants expressing wild type CD26 has greater sensitivity to doxorubicin and etoposidemediated G₂ arrest than parental cells or transfectants expressing CD26 mutated at the DPPIV site, which is associated with enhanced topoisomerase II alpha expression. Evaluating CD26 expression on subsets of T cell malignancies, we found that the absence of CD26 expression is a useful marker for the diagnosis of Mycosis Fungoides/Sezary Syndrome involvement in the peripheral blood. Furthermore, CD26 may be a marker of aggressive T-large granular lymphocyte lymphoproliferative disorder, and its ability to conduct T cell costimulatory signals may be aberrant in this disease. Lastly, our clinical trial suggested that the ADA inhibitor pentostatin may preferentially target CD26+ T lymphocytes, with potential clinical implications. (Presented at the 1233rd Meeting, October 19, 2001.)

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