Decreased TLR4 gene expression in leukemic leukocyte populations.

Toll-like receptor 4 (TLR4) is one member of a class of pattern recognition receptors that play a significant role in the physiologic innate immune response. As leukemia is a disease state that may be associated with a compromised immune system, it was hypothesized that depressed TLR4 function resulting from decreased gene expression might be related to the development and further sustained presence of a leukemic clone of cells. This study thus analyzed gene expression of TLR4 in groups of lymphocytic leukemia cases, myeloid leukemia cases, cases of myeloid leukemia in remission, and normal controls by real-time quantitative reverse transcription-PCR (qRT-PCR). It was observed that TLR4 gene expression was indeed decreased to a statistically significant degree (P<0.05) in both the lymphocytic leukemic subset and myeloid leukemic subset when compared to normal controls. Thus, further study is warranted into determining whether this decreased TLR4 expression contributes to the pathogenesis of leukemic clone development through an associated depressed immune surveillance as well as whether TLR4 agonists might serve to effectively strengthen the response of the immune system in battling leukemic burden.

Cellular and molecular alterations in spinal cord injury patients with pressure ulcers: a preliminary report.

The study was designed to investigate the changes, both numerically and functionally, of the molecules critical to wound healing in spinal cord injury (SCI) patients. Spinal cord injury patients who demonstrated delayed healing of their pressure ulcers were used as study subjects. Age-matched healthy individuals served as controls. Adhesion molecule expression of the peripheral blood leukocytes, including lymphocytes and granulocytes, was measured by flow cytometric analysis. Binding capacity of the lymphocytes was evaluated using human umbilical cord vein endothelial cells (HUVECs) as the binding matrix. Samples from pressure ulcers of the patients were immunostained to define fibronectin, kalinin, beta4 integrin, alpha2beta1, alpha3beta1, alpha5beta1, and CD138 expression. Compared to healthy controls, there was decreased expression of CD11a, CD11b, CD18, CD49b, CD49c, CD49d, CD54, and CD8 in patients' lymphocyte populations and CD11a, CD18, CD49c, CD49d, and CD8 in patients' granulocyte populations. The binding capacity, expressed as percentage binding of the lymphocytes to the HUVEC matrix, was greatly diminished in the patients. There was markedly diminished immunohistochemical staining of fibronectin in pressure ulcers. These findings showed that delayed healing of pressure ulcers in SCI patients can be attributed to reduced adhesion molecule expression, impaired cell-cell interaction, and lack of extracellular matrix structural and functional protein.

High panel reactive antibody against cross-reactive group antigens as a contraindication to renal allotransplantation.

Preformed circulating cytotoxic IgG anti-HLA alloantibodies induced by previous failed grafts, blood transfusion, or pregnancy are a contraindication to allotransplantation and result in hyperacute rejection. These persistent, highly cytotoxic panel reactive antibodies (PRAs) may be specific for epitopes that are shared among HLA antigens known as cross-reactive groups (CREGs). The present investigation includes 24 subjects awaiting renal transplants with flow cytometric PRAs >30%. Eighty-seven percent of the patients developed alloantibodies specific for the mismatched antigens of previous failed grafts. The complement-dependent cytotoxicity test revealed that A1 and A2 antigens were highly immunogenic, whereas A23, B35, and B7 were less so. All patients who formed anti-A1 and anti-A2 also had developed alloantibodies specific for other antigens of the 1C and 2C CREGs, respectively. The presence of anti-class II HLA alloantibodies led to poor graft survival, i.e., a maximum of 2 years.

Fate of immune complexes, glomerulonephritis, and cell-mediated vasculitis in lupus-prone MRL/Mp lpr/lpr mice.

Immune complex formation was induced by the injection of (125)I-BSA into female MRL/Mp lpr/lpr mice, which develop spontaneous systemic lupus erythematosus (SLE)-like disease, and MRL/Mp +/+ mice, which do not. At designated intervals following the injection of 10 mg of (125)I-bovine serum albumin (BSA), the nonlupus mice developed sparse, small electron-dense deposits in mesangial areas and subepithelial immune deposits that underwent partial resolution. By contrast, glomeruli of the SLE-prone mouse kidneys revealed proliferation of mesangial cells and some increase in mesangial matrix material. Numerous subepithelial and mesangial electron-dense deposits were present. Some subendothelial and intramembranous deposits were also demonstrated. Capillary lumens contained massive electron-dense deposits. The resolving subepithelial deposits observed were fewer than half the number found in kidneys of the non-SLE mice. Whole body counts were also recorded daily following the injection of (125)I-BSA. Whereas, both lupus-prone and non-SLE control mice eliminated (125)I-BSA at equivalent rates through day 12 postinoculation, those with SLE-like disease showed a decreased (125)I-BSA elimination rate between days 6 and 12. Results suggest an impairment in the ability of SLE-prone mice to resolve immune complexes, whether they are nuclear-antinuclear or from an exogenous source, i.e., BSA-anti-BSA, compared to controls in this experimental model of the superimposition of exogenous immune complex formation on systemic lupus erythematosus-like disease.

Review of immune function, healing of pressure ulcers, and nutritional status in patients with spinal cord injury.

The immune, neural, and endocrine systems do not act autonomously; rather, multiple communicative pathways exist among the nervous, endocrine, and immune systems that facilitate physiological immunoregulation. Patients with spinal cord injury (SCI) have decreased natural and adaptive immune responses by 2 weeks after injury. In patients with SCI, adrenocorticotropic hormone (ACTH) and urine-free cortisol levels were increased while zinc and albumin levels were decreased, respectively. In addition, the surface markers alpha 2, alpha 3, alpha 4, CD11a, CD11b, CD18, CD54, and CD8 found on lymphocytes and alpha 3, alpha 4, CD11a, CD18, and CD8 surface markers found on granulocytes were also decreased in the patient population. Finally, the adhesion molecules binding ability in the SCI group was also decreased when compared with a control group. Overall, the investigation showed that patients with SCI have a decreased immune function, especially succeeding the SCI injury, an impaired nutrition status, and a decreased number of adhesion molecules, all of which contribute to delayed wound healing.

Association of HLA-DQ and -DR alleles with protection from or infection with HIV-1.

Infection with human immunodeficiency virus type 1 (HIV-1) and progression to acquired immune deficiency syndrome (AIDS) are controlled by both host genetic factors and viral factors. The HLA (human leukocyte antigen) region in humans controls immune response functions and tissue rejection and influences susceptibility to neoplasia, autoimmune diseases, and infectious diseases including HIV. Twenty-eight African American and 12 Caucasian patients participated in the study. HLA-DQB1 and HLA-DRB1 genotyping was performed using PCR and sequence-specific oligonucleotide probe reverse hybridization and analyzed with the LiPA Key Typing System and LiPA software. DQB1*0603 was found to be positively associated with HIV-1 infection and with HIV-1 infection in Caucasians but not African Americans. DQB1*03032 frequencies indicate a positive association with protection from HIV-1 infection. It was further found to be protective against HIV-1 infection in Caucasians but not in African Amens. DQB1*0201 was observed more frequently in HIV(+) African Americans than HIV(-) African Americans, suggesting a positive association with HIV-1 infection in this ethnic group. HLA-DRB1*04 exhibited a positive association with HIV-1 infection in Caucasians. These data show that there are HLA class II alleles associated with susceptibility to and protection from HIV-1 infection and that these differ between ethnic groups.

Facilitation of immune function, healing of pressure ulcers, and nutritional status in spinal cord injury patients.

Multiple communicative pathways among nervous, endocrine, and immune systems facilitate physiological immunoregulation. Spinal cord injury (SCI) patients had strikingly decreased natural and adaptive immune responses by 2 weeks post injury. While NK-cell function was decreased, plasma ACTH and urine-free cortisol levels were increased. T cell function and activation were both diminished. With rehabilitation therapy, NK and T function increased; without rehabilitation, NK levels remained depressed. When rehabilitation ceased, NK function decreased. Cervical SCI patients had less NK and T function than thoracic injury patients. SCI patients also had reduced levels of cellular adhesion molecules (CAMs) that participate in immune function and wound healing. SCI patients with pressure ulcers were compared to those without pressure ulcers. LFA-1, VLA-4, and other surface markers were decreased on the lymphocytes of all SCI patients. SCI patients with pressure ulcers had lower CAM levels than did patients without pressure ulcers. Nutritional status was determined by zinc, albumin, and prealbumin levels. SCI patients had decreased albumin levels. Those with pressure ulcers had decreased prealbumin levels and zinc levels.

History of medicine: the metamorphosis of scientific medicine in the ever-present past.

Hippocrates (460-370 BCE), the father of medicine, developed principles for medical diagnosis and treatment together with a code of ethics. When the first Ptolemy ruled Egypt, he created a great library of 700,000 rolls at Alexandria, which became a repository for the works of Socrates, Plato, Aristotle, Hippocrates, and all the writings of the known world, but it was destroyed by a great fire. Galen of Pergamum (129-216), who lived 500 years after Hippocrates, was well educated and studied anatomy, surgery, drugs and Hippocratic medicine. His ideas influenced medical thinking for the next 1500 years. The Arabic physician Ibn Sina (Avicenna) wrote a great medical work entitled Canon of Medicine. After the Dark Ages (500 to 1050), academic medicine was reestablished in Europe, especially at Salerno, Bologna, Padua, Paris, Montpellier, and Oxford. The greatest medical disaster of the Middle Ages was the Black Death. Other diseases of note were leprosy, smallpox, tuberculosis, typhus, measles, diarrhea, meningitis, and colic. As interest in human dissection increased, the study of anatomy became popular. With development of the printing press, medical knowledge became more widely disseminated and technical advances in science flourished. Advances in medicine occurred in concert with developments in technology. These included the microscope, the stethoscope, anesthetic agents, discoveries in bacteriology, a carbolic acid spray to reduce infection during surgery, the clinical thermometer, blood transfusions, electrocardiography, X-rays, and the sphygmomanometer. Johns Hopkins University was established at the end of the 19th century to train scientifically knowledgeable physicians. The first faculty included Welch, Osler, Halstead, Kelly, Mall, and Abel. Graduates of the new school carried scientific medicine to universities throughout America. More medical advances have been made during the 20th century than in all the other centuries combined. Advances in medical knowledge have resulted not only from developments in technology but from increased access to current information provided through libraries such as the National Library of Medicine in Bethesda, Maryland.

HLA-DQB1 markers associated with human immunodeficiency virus type I disease progression.

In a previous investigation, we demonstrated that certain human leukocyte antigens (HLA) may be associated with human immunodeficiency virus type I (HIV-1) infection or protection from infection among regional African Americans and Caucasians. We demonstrated that HLA-DQB1*0605 was associated with a possible increased risk of susceptibility to infection in African Americans and that DQB1*0602 was associated with a possible increased risk of infection in Caucasians. The present study was designed to demonstrate possible HLA associations with HIV-1 disease progression and AIDS in regional African American and Caucasian populations. To differentiate rapid from slow progressors, immune parameters of the HIV-1-positive patient population were monitored over a mean follow-up period of 23 +/- 2 months for African Americans (n = 30) and 25 +/- 5 months for Caucasians (n = 22). To determine significance, HLA allele frequencies among rapid progressors were compared to those of slow progressors, separated by race. Results were analyzed by chi 2 analysis, with Fisher's exact test where applicable, linear logistic regression and Kaplan-Meier survival analysis. In the HIV-1-positive African American group, a better prognosis was associated with HLA-DQB1*0602. In the HIV-1-positive Caucasian group, HLA-DQB1*0302 was associated with rapid HIV disease progression, but no marker was associated with a more favorable prognosis.

Restoration of depressed immune function in spinal cord injury patients receiving rehabilitation therapy.

Both natural and adaptive immune responses were strikingly decreased 2 weeks after injury in 49 spinal cord injuries, 28 tetraplegic and 21 paraplegic patients compared to agematched controls. All values are expressed as means. NK cell function decreased to 21.0% 2 weeks after spinal cord injury compared to 48.6% in controls. At 2 weeks, plasma ACTH values increased to 17.0 pg/ml in patients compared to 11.2 pg/ml in controls and urine free cortisol levels were elevated to 162.4 micrograms/24 h in patients compared to 53.6 ug/24 h in controls. T cell function decreased to 40.2% of normal (lymphocyte transformation) by 3 months post injury. T cell activation (IL-2R) was diminished, i.e., 183.4 ug/ml compared to 328.2 ug/ml in controls. With rehabilitation therapy, NK cell function increased to 41.6% by 7 months post injury. NK cell-mediated lysis diminished sharply between 7 and 9 months decreasing to 22.8% at 10 months and ultimately returning to the 2 week post injury level. Rehabilitation therapy contributed to the restoration of T cell function to 92.0% of normal by 6 months post injury where it remained for 6+ months. IL-2R values improved in parallel with lymphocyte transformation. Whereas NK cell-induced lysis remained depressed, i.e., 11.8% at 6 months and 11.4% at 12+ months in patients not receiving therapy, the restoration of NK cell function at 6 months to 40.6% in rehabilitated patients decreased to 23.0% with cessation of treatment. NK cell-mediated lysis values in cervical injury patients were significantly less than those in the thoracic injury group. FIM scores of the two paralleled their NK cell function. With rehabilitation therapy, NK cell-mediated lysis in the cervical group increased from 15.2% to 28.4%, whereas it improved in the thoracic group with therapy from 26.8% to 43.7%. With rehabilitation therapy, lymphocyte transformation in the cervical group increased from 37.3% to 85.6% and improved in the thoracic group from 48.4% to 88.9%. With rehabilitation therapy, FIM scores improved from 49.7 to 74.0 in the cervical group and from 79.8 to 97.3 in thoracic patients compared to 126 in controls of healthy age matched controls.

Adhesion molecules and wound healing in spinal cord injury.

The purpose of this study was to design and test a model that could identify and define which cellular adhesion molecules (CAMs) present on peripheral blood leukocytes were depressed in spinal cord injury (SCI) patients. CAMs on peripheral blood cells of SCI patients with pressure ulcers were measured by flow cytometry and compared with those of age-matched healthy controls and SCI patients on physical rehabilitation therapy (PRT) protocols without pressure ulcers. The latter patients had normal levels (97%) of lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) and a low incidence of infection. By contrast, prior to undergoing rehabilitation therapy, SCI patients with pressure ulcers had significantly diminished LFA-1 levels (62%). Very late antigen 4 (VLA-4; alpha 4 beta 1; 34%) levels (i.e., alpha 4 = 34% and beta 1 = 44%) were approximately half those in controls (72%). Expression of alpha 2 and alpha 3 was also diminished in patients. Patients receiving PRT after debridement developed increased levels of LFA-1 and VLA-4 by the 6th week but alpha 2 and alpha 3 remained relatively low. These results combined with data from previous studies suggest that patients not receiving PRT developed severe pressure ulcers which required debridement surgery and healed more slowly due, in part, to reduced levels of CAMs.

HIV-1 disease association with HLA-DQ antigens in African Americans and Caucasians.

Previously, we have shown that CD4 levels in African Americans infected with human immunodeficiency virus-1 (HIV-1) were lower than those in Caucasians. To determine whether or not HLA type is associated with susceptibility to HIV-1 infection, we demonstrated serologically that HLA-DQ6(1) and HLA-DQ7(3) were associated with HIV-1 infection in both African Americans and Caucasians. The present investigation was designed to demonstrate whether or not HLA-DQB1 alleles were associated with HIV-1 infection or protection from infection within these two ethnic groups. Oligonucleotide typing was employed and results were analyzed by chi 2 with Fisher's exact test to compare HLA-DQ marker frequencies in the regional control population (98 African Americans, 143 Caucasians) to the disease population (n = 52; 30 African Americans and 22 Caucasians). We found a statistically significant increased risk of HIV infection associated with HLA-DQB1*0605 in African Americans, and with HLA-DQB1*0602 in Caucasians. By contrast, HLA-DQB1*0603 was associated with protection in Caucasians.

Immune system-neuroendocrine dysregulation in spinal cord injury.

Multiple communicative pathways among the nervous, endocrine and immune systems facilitate physiological immunoregulation. Spinal cord injury (SCI) patients have decreased natural (NK cell) and adaptive (T cell) immune function and reduced blood levels of cellular adhesion molecules (CAMs) that participate in immune function and wound healing. We found decreased LFA-1 and VLA-4 on peripheral blood leukocytes in SCI patients and lower levels of CAMs in SCI patients with pressure ulcers than in those without them. SCI might affect immune cells and immune responsiveness by: (1) disrupting the outflow of signals from the sympathetic nervous system to lymphoid tissues and their blood vessels as well as the returning afferent signals from these tissues to the brain; (2) immunosuppression caused by the stressors affecting SCI patients; (3) interrupting returning signals to the CNS from the periphery thereby reducing facilitation of immunoregulatory CNS neurons and decreasing their activity; or a combination of all three. SCI patients may develop dysregulation of the sympathetic nervous system that is intimately involved in immune function. Chronic stress mediates immunosuppression by corticosteroids, catecholamines, endorphins and met-enkephalin. The hypothalamus coordinates the response to stress through the release of soluble products from the sympathetic nervous system and hypothalamic-pituitary-adrenal axis. Whereas the nervous and endocrine systems are not concerned with immunological specificity, they do influence the intensity, kinetics and localization of immune responses. Products of an activated immune system may generate feedback circuits capable of inhibiting, enhancing or regulating neuronal input. Immune system cells can produce neurologically active peptides including ACTH, CRF, growth hormone, thyrotropin, prolactin, human chorionic gonadotropin, endorphin, enkephalins, substance P, somatostatin and VIP. Cytokines are likely important mediators of the HPA response to immune stimuli.

Progression of HIV infection is associated with HLA-DQ antigens in Caucasians and African Americans.

In our previous work with human leukocyte antigen (HLA) association in human immunodeficiency virus (HIV) infection, African Americans (Afr Ams) and Caucasians (Caucs) exhibited HLA markers that were associated with protection or disease. The present study was designed to establish if HLAs were associated with the severity of HIV infection and progression to AIDS in Afr Am and Cauc adults. The frequency of serologically determined antigens (Ags) in the regional control population was compared to the HIV-infected population and the HIV-infected slow progressors were compared to rapid progressors by race. chi 2 analysis with Bonferroni adjustment, Kaplan-Meier survival analysis, linear logistic regression, Cox model of proportional hazards and standardized deltas were applied as applicable. Immune parameters were monitored over a mean follow-up period of 23 +/- 2 months for Afr Ams (n = 35) and 25 +/- 5 months for Caucs (n = 24). A better prognosis in the HIV+Afr Am group was associated with HLA-DQ1 with a risk ratio of 0.295. In the HIV+Cauc group, a preferable prognosis was associated with HLA-DQ3 with a risk ratio of 0.11, and a poor prognosis was associated with HLA-DQ2 with a risk ratio of 7. Afr Am haplotypes that appeared to have the greatest association with rapid progression of HIV infection were A69(28)-B40 and related haplotypes as well as B12-DR14(6). Cauc haplotypes with the strongest association with rapid and slow progression of HIV infection were A28-B17-DR9 and A30(19)-B67, respectively. The DR Ags of at least one haplotype that led to rapid progression in both races were associated with DQ9(3). An 'immune response' gene (DQ region) may control the progression of HIV infection in adults. The rapidly progressive DQ-associated peptide might block the progression of HIV if given as a novel vaccine.

The immune system victorious: selective preservation of self.

How the body successfully distinguishes its own tissue cells from those that are foreign and genetically nonidentical to it has been a focus of much research. Clonal deletion maintains that immune system cells with the potential to injure self constituents are eliminated during development, thereby neutralizing their capacity to induce self injury. Selected self-reactive maturing T cell clones undergo deletion in the thymus. A two-step selection process affects immature T cells that enter the thymus. Positive selection makes certain that all surviving cells are able to identify major histocompatibility complex (MHC) proteins present on all body cells. These MHC proteins interact with antigens and present them to T lymphocytes. Negative selection is essential for self-tolerance. It eliminates potentially injurious self-reactive T cells by placing them in contact with a mixture of self antigens in the thymus. Clonal anergy might act together with clonal deletion to maintain self tolerance. Self-reactive T cells in the blood of healthy subjects could represent cells whose affinities for antigen are too weak to initiate an immunologic disease. The fate of T cells reacting to a specific antigen has been traced in transgenic mice. Class I MHC molecules present peptides manufactured within the cell, whereas class II MHC molecules present peptides from extracellular proteins. Interaction of a T cell receptor with its homologous antigen associated with MHC molecules leads to proliferation of that T cell in the presence of costimulatory signals. Investigations elucidating the role of T cell receptors, MHC molecules and antigen peptides in self-nonself discrimination are discussed. The article concludes with an introductory summary of the remaining articles in the issue that address selected topics in self-nonself discrimination.

Decreased immune reactivity and neuroendocrine alterations related to chronic stress in spinal cord injury and stroke patients.

Both natural and adaptive immune responses were found to be strikingly decreased 2 weeks after injury in 54 spinal cord injury and stroke patients, i.e., 28 quadriplegics, 21 paraplegics and 5 stroke patients, compared with those of age-matched controls. All values are expressed as means. Natural-killer (NK)-cell function decreased to 21.0% 2 weeks after spinal cord injury compared with 48.6% in controls. At 2 weeks, plasma ACTH values increased to 17.0 pg/ml in patients compared with 11.2 pg/ml in controls, and urine free cortisol levels were elevated to 162.4 micrograms/24 h in patients compared with 53.6 micrograms/24 h in controls. T-cell function decreased to 40.2% of normal (lymphocyte transformation) by 3 months after injury. T-cell activation (IL-2R) was diminished, i.e., 183.4 micrograms/ml compared with 328.2 micrograms/ml in controls. With rehabilitation therapy, NK-cell function increased to 41.6% by 7 months after injury. NK-cell-mediated lysis diminished sharply between 7 and 9 months, decreasing to 22.8% at 10 months and ultimately returning to the level seen 2 weeks after injury. Rehabilitation therapy contributed to the restoration of T-cell function to 92.0% of normal by 6 months after injury where it remained for 6+ months. IL-2R values improved in parallel with lymphocyte transformation. Whereas NK-cell-induced lysis remained depressed, i.e., 11.8% at 6 months and 11.4% at 12+ months in patients not receiving therapy, the restoration of NK-cell function at 6 months to 40.6% in rehabilitated patients decreased to 23.0% with cessation of treatment. NK-cell-mediated lysis values in cervical injury patients were significantly less than those in the thoracic injury group. Functional independence measurement (FIM) scores of the two paralleled their NK-cell function. With rehabilitation therapy, NK-cell-mediated lysis in the cervical group increased from 15.2 to 28.4%, whereas it improved in the thoracic group with therapy from 26.8 to 43.7%. With rehabilitation therapy, lymphocyte transformation in the cervical group increased from 37.3 to 85.6% and improved in the thoracic group from 48.4 to 88.9%. With rehabilitation therapy, FIM scores improved from 49.7 to 74.0 in the cervical group and from 79.8 to 97.3 in thoracic patients compared with 126 in controls. NK-cell-mediated lysis was depressed to 28.9% in 5 stroke patients and improved to 38.0% following rehabilitation therapy.

Antithyroid and goitrogenic effects of coal-water extracts from iodine-sufficient goiter areas.

Goiter in iodine-sufficient areas has been linked to water-borne goitrogens in watersheds and aquifers rich in coal and shale. In the present study, the potential antithyroid and goitrogenic effects of coal-water extracts (CWE) were investigated in vivo in rats after chronic and acute oral administration of CWE, and in vitro by a thyroid peroxidase (TPO) enzyme system. CWE was prepared by continuous extraction of ground (40 mesh) Appalachian coal with goitrogen-free water (GFW). Female Buffalo rats fed on Purina iodine-rich diet (12 micrograms I-/day/rat), were given ad lib CWE (50 mg/ml; approximately 20 mL/day/rat) or GFW (controls) for 2 months. At the end of the experiment, 125I 1 microCi, was injected i.p. and 4 h later the thyroid glands were removed, weighed, and analyzed histologically and for total 125I and 125I-labeled compounds. Rats on CWE had larger thyroid glands [7.2 +/- 0.3 mg/100 g (mean +/- SE) vs 5.0 +/- 0.5 controls; p < 0.005] with distinct histological changes of smaller thyroid follicles, some with columnar epithelium, and with more dense colloid than in controls, and had significant inhibition of the coupling mechanism for production of thyroid hormones [125MIT + DIT/125T3 + T4: 5.1 +/- 0.2 vs 3.9 +/- 0.1 controls, p < 0.005; and 125T3 + T4 (%): 10.6 +/- 0.3 vs 12.6 +/- 0.4 controls, p < 0.005]. Female Sprague-Dawley rats under the same conditions as Buffalo rats were given acutely by GI tube 2 mL of CWE (5 g/mL) or GFW (controls).(ABSTRACT TRUNCATED AT 250 WORDS)