Aggrecanolysis in human osteoarthritis: confocal localization and biochemical characterization of ADAMTS5-hyaluronan complexes in articular cartilages.

OBJECTIVE: Human osteoarthritis (OA) is characterized by aggrecanase-mediated depletion of cartilage aggrecan. We have examined the abundance, location and some biochemical properties of the six known aggrecanases (A disintegrin and metalloproteinase with thrombospondin-like motifs 1 (ADAMTS1) 4, 5, 8, 9 and 15) in normal and OA human cartilages. METHODS: Formalin-fixed, ethylenediamine tetraacetic acid (EDTA)-decalcified sections of full-depth cartilage from human OA tibial plateaus and normal control samples were studied by confocal imaging. Probes included specific antibodies to aggrecanases and two aggrecan epitopes, as well as biotinylated hyaluronan binding protein (HABP) for hyaluronan (HA) visualization. Cartilage extracts were analyzed by Western blot for the individual proteinases and aggrecan fragments. RESULTS: ADAMTS5 was present in association with cells throughout normal cartilage and was markedly increased in OA, particularly in clonal groups in the superficial and transitional zones, where it was predominantly co-localized with HA. Consistent with the confocal analysis, a high molecular weight complex of ADAMTS5 and HA was isolated from human OA cartilage by isotonic salt extraction and chromatography on Superose 6. The complex eluted with an apparent molecular size of about 2x10(6) and contained major ADAMTS5 forms of 150, 60, 40 and 30kDa. The yield of most forms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was markedly enhanced by prior digestion of the complex with either Streptomyces hyaluronidase or chondroitinase ABC. CONCLUSION: ADAMTS5 abundance and distribution in human OA cartilages is consistent with a central role for this enzyme in destructive aggrecanolysis. HA-dependent sequestration of ADAMTS5 in the pericellular matrix may be a mechanism for regulating the activity of this proteinase in human OA cartilage.

Mortality among a cohort of uranium mill workers: an update.

AIMS: To evaluate the mortality experience of 1484 men employed in seven uranium mills in the Colorado Plateau for at least one year on or after 1 January 1940. METHODS: Vital status was updated through 1998, and life table analyses were conducted. RESULTS: Mortality from all causes and all cancers was less than expected based on US mortality rates. A statistically significant increase in non-malignant respiratory disease mortality and non-significant increases in mortality from lymphatic and haematopoietic malignancies other than leukaemia, lung cancer, and chronic renal disease were observed. The excess in lymphatic and haematopoietic cancer mortality was due to an increase in mortality from lymphosarcoma and reticulosarcoma and Hodgkin's disease. Within the category of non-malignant respiratory disease, mortality from emphysema and pneumoconioses and other respiratory disease was increased. Mortality from lung cancer and emphysema was higher among workers hired prior to 1955 when exposures to uranium, silica, and vanadium were presumably higher. Mortality from these causes of death did not increase with employment duration. CONCLUSIONS: Although the observed excesses were consistent with our a priori hypotheses, positive trends with employment duration were not observed. Limitations included the small cohort size and limited power to detect a moderately increased risk for some outcomes of interest, the inability to estimate individual exposures, and the lack of smoking data. Because of these limitations, firm conclusions about the relation of the observed excesses in mortality and mill exposures are not possible.

Inhibition of NFkappaB induces caspase-independent cell death in human T lymphocytes.

Nuclear factor kappaB (NFkappaB) regulates the expression of various genes essential for cell survival. Here we demonstrate that suppression of NFkappaB nuclear import with SN50 peptide carrying the nuclear localization sequence (NLS) of the NFkappaB p50 subunit induces apoptosis in human peripheral blood T lymphocytes (T-PBL), which can be blocked with the pan-caspase inhibitor Z-VAD.fmk. However, even when caspase function is blocked, the addition of SN50 induces irreversible cell loss due to the reduction in the mitochondrial transmembrane potential (DeltaPsim) followed by disruption of the cell membrane, hallmarks of necrosis. These observations demonstrate that although inhibition of NFkappaB nuclear translocation by SN50 peptide can induce caspase-dependent apoptosis in T-PBL, cell death may still proceed in the absence of functional caspase activity. The availability of downstream caspases appears to determine the mode of cell death in NFkappaB defective cells.

The costimulatory molecule ICOS plays an important role in the immunopathogenesis of EAE.

The inducible costimulatory molecule (ICOS) is expressed on activated T cells and participates in a variety of important immunoregulatory functions. After the induction of experimental allergic encephalomyelitis in SJL mice with proteolipid protein (PLP), brain ICOS mRNA and protein were up-regulated on infiltrating CD3+ T cells before disease onset. ICOS blockade during the efferent immune response (9-20 days after immunization) abrogated disease, but blockade during antigen priming (1-10 days after immunization) exacerbated disease. Upon culture with PLP and compared with immunized controls, splenocytes produced either decreased interferon-gamma (IFN-gamma, in efferent blockade) or excessive IFN-gamma (in priming blockade). PLP-specific immunoglobulin G1 was decreased in animals treated with anti-ICOS during antigen priming, but not in other groups.

Workshop on harmonization of serving future needs with occupational exposure databases--inhalation modeling, session IIIA.

This workshop was one of several that took place at the International Symposium on Occupational Exposure Databases and Their Application for the Next Millennium held in London from November 1-3, 1999. About 30 delegates participated in the workshop. The agenda for the discussions was provided by a white paper prepared by the organizers. The workshop produced a conceptual outline for a general-purpose prediction model for inhalation exposure, and constructed a list of important input variables for successful model development. Evaluation of prototype models was discussed in some detail, and the workshop concluded with suggestions for taking forward the ideas discussed and maintaining the momentum and interest generated during the symposium.

The NIOSH/FAA Working Women's Health Study: evaluation of the cosmic-radiation exposures of flight attendants. Federal Aviation Administration.

Air crew are exposed to elevated levels of cosmic ionizing radiation of galactic and solar origin and are among the more highly exposed occupational groups to ionizing radiation in the United States. Depending on flight route patterns, the annual dose may range from 0.2 to 5 mSv. By comparison, the average annual radiation dose equivalent of occupationally exposed adults in the United States is estimated to be 1.1 mSv. Cosmic-radiation dose depends primarily on altitude and geomagnetic latitude and to a lesser degree on solar activity. Although the International Commission on Radiological Protection has recommended that air crew exposures to natural radiation in-flight be treated as occupational exposures, United States flight crew exposures to natural cosmic radiation are not regulated or typically monitored. There are approximately 148,000 air crew (flight deck crew and flight attendants) in the United States.

Healing of diabetic foot ulcers and pressure ulcers with human skin equivalent: a new paradigm in wound healing.

HYPOTHESIS: In patients with diabetic foot and pressure ulcers, early intervention with biological therapy will either halt progression or result in rapid healing of these chronic wounds. DESIGN: In a prospective nonrandomized case series, 23 consecutive patients were treated with human skin equivalent (HSE) after excisional debridement of their wounds. SETTING: A single university teaching hospital and tertiary care center. PATIENTS AND METHODS: Twenty-three consecutive patients with a total of 41 wounds (1.0-7.5 cm in diameter) were treated with placement of HSE after sharp excisional debridement. All patients with pressure ulcers received alternating air therapy with zero-pressure alternating air mattresses. MAIN OUTCOME MEASURE: Time to 100% healing, as defined by full epithelialization of the wound and by no drainage from the site. RESULTS: Seven of 10 patients with diabetic foot ulcers had complete healing of all wounds. In these patients 17 of 20 wounds healed in an average of 42 days. Seven of 13 patients with pressure ulcers had complete healing of all wounds. In patients with pressure ulcers, 13 of 21 wounds healed in an average of 29 days. All wounds that did not heal in this series occurred in patients who had an additional stage IV ulcer or a wound with exposed bone. Twenty-nine of 30 wounds that healed did so after a single application of the HSE. CONCLUSIONS: In diabetic ulcers and pressure ulcers of various durations, the application of HSE with the surgical principles used in a traditional skin graft is successful in producing healing. The high success rate with complete closure in these various types of wounds suggests that HSE may function as a reservoir of growth factors that also stimulate wound contraction and epithelialization. If a wound has not fully healed after 6 weeks, a second application of HSE should be used. If the wound is not healing, an occult infection is the likely cause. All nonischemic diabetic foot and pressure ulcers that are identified and treated early with aggressive therapy (including antibiotics, off-loading of pressure, and biological therapy) will not progress.

Tumor-induced dysfunction in interleukin-2 production and interleukin-2 receptor signaling: a mechanism of immune escape.

PURPOSE: The development of an effective antitumor immune response is compromised in patients with renal cell carcinoma. Despite significant infiltration by T lymphocytes into renal tumors, no detectable induction of gene expression is associated with the generation of an antitumor immune response. Tumor-induced down-regulation of interleukin (IL)-2 expression may contribute to the impaired development of the T cell-mediated antitumor immune response. Within renal tumors, there is no detectable expression of IL-2 or the IL-2 receptor alpha chain, and only low levels of interferon gamma (IFN-gamma) mRNA are detected. Products in the tumor environment may suppress the expression of these genes, thus inhibiting production of type 1 helper T cell cytokines. METHODS: Peripheral blood lymphocytes obtained from healthy volunteers were exposed to supernatants from renal cell carcinoma explants, and the immunologic consequences of this were assessed using a variety of molecular assays. RESULTS: Soluble products from renal tumor explants can inhibit the production of IL-2 and IFN-gamma by peripheral blood lymphocytes and can suppress T-cell proliferation. Soluble products from renal cell carcinoma explants appear to block the nuclear translocation of nuclear factor kappa B (NFkappaB) proteins p50 and RelA without affecting cytoplasmic levels of these proteins. In some experiments, a reduction in the nuclear translocation of other transcription factors involved in IL-2 gene expression, including nuclear factor of activated T cells and accessory protein-1, was observed. Gangliosides isolated from tumor supernatants blocked the production of IL-2 and IFN-gamma in response to ionomycin plus phorbol myristate acetate stimulation. These gangliosides also inhibited stimulus-dependent activation and nuclear accumulation of NFkappaB. Coculture experiments demonstrated that renal cell carcinoma lines known to express gangliosides could inhibit the activation of NFkappaB in normal T cells and the Jurkat T-cell line. Supernatants from renal cell carcinoma explants and renal cell carcinoma cell lines can also suppress the proliferation of normal T cells, thus reproducing another defect observed in tumor-infiltrating lymphocytes. Supernatants from renal cell carcinoma tumors also appear to inhibit signaling through the IL-2 receptor. Although tumor supernatants had little effect on IL-2 receptor (alpha, beta or gamma) expression, they did block expression of JAK3, a key kinase involved in signaling through the IL-2 receptor pathway. Moreover, downstream events in IL-2 receptor signaling linked to JAK3 were impaired in T cells treated with tumor supernatants. CONCLUSION: These findings suggest that soluble products from renal tumors may suppress T-cell responses by blocking both IL-2 production and normal IL-2 receptor signaling.

Renal cell carcinoma-derived gangliosides suppress nuclear factor-kappaB activation in T cells.

Activation of the transcription factor nuclear factor-kappaB (NFkappaB) is impaired in T cells from patients with renal cell carcinomas (RCCs). In circulating T cells from a subset of patients with RCCs, the suppression of NFkappaB binding activity is downstream from the stimulus-induced degradation of the cytoplasmic factor IkappaBalpha. Tumor-derived soluble products from cultured RCC explants inhibit NFkappaB activity in T cells from healthy volunteers, despite a normal level of stimulus-induced IkappaBalpha degradation in these cells. The inhibitory agent has several features characteristic of a ganglioside, including sensitivity to neuraminidase but not protease treatment; hydrophobicity; and molecular weight less than 3 kDa. Indeed, we detected gangliosides in supernatants from RCC explants and not from adjacent normal kidney tissue. Gangliosides prepared from RCC supernatants, as well as the purified bovine gangliosides G(m1) and G(d1a), suppressed NFkappaB binding activity in T cells and reduced expression of the cytokines IL-2 and IFN-gamma. Taken together, our findings suggest that tumor-derived gangliosides may blunt antitumor immune responses in patients with RCCs.

Evaluation of exposures to fluorocarbon 113 in a horizontal and a vertical laminar airflow clean room.

Exposures to 1,1,2-trichloro-1,2,2-trifluoroethane or fluorocarbon (FC) 113 were evaluated in a horizontal laminar airflow (HLAF) clean room and a vertical laminar airflow (VLAF) clean room. A full period consecutive samples measurement strategy was employed. Data were used to calculate 8-hour time-weighted averages (8-TWA) for major work groups and to characterize exposures associated with specific cleaning tasks. The MIRAN 1B infrared analyzer was used to estimate peak concentrations. In the HLAF clean room, 8-TWAs ranged from 193 to 439 ppm; in the VLAF clean room, 8-TWAs ranged from 110 to 935 ppm. These levels were below the current Occupational Safety and Health Administration permissible exposure limit and the National Institute for Occupational Safety and Health (NIOSH) recommended exposure limit for FC 113 of 1000 ppm. Short-term sample concentrations ranged from 104 ppm (inspection) to 1080 ppm (assembly) in the HLAF clean room and 51 ppm (packaging)-3380 ppm (flushing) in the VLAF clean room. In the VLAF clean room, several short-term concentrations measured during the flushing task--1421 ppm and 2522 ppm--were above the NIOSH short-term exposure limit (STEL) of 1250 ppm. These data suggest the possibility that the STEL may be exceeded for tasks involving direct work with liquid FC 113. Peak exposure levels may be reduced by modification of worker position in the HLAF clean room and by use of open wire tables in the VLAF clean room.

Inhibition of NF-kappa B activity in human T lymphocytes induces caspase-dependent apoptosis without detectable activation of caspase-1 and -3.

NF-kappa B is involved in the transcriptional control of various genes that act as extrinsic and intrinsic survival factors for T cells. Our findings show that suppression of NF-kappa B activity with cell-permeable SN50 peptide, which masks the nuclear localization sequence of NF-kappa B1 dimers and prevents their nuclear localization, induces apoptosis in resting normal human PBL. Inhibition of NF-kappa B resulted in the externalization of phosphatidylserine, induction of DNA breaks, and morphological changes consistent with apoptosis. DNA fragmentation was efficiently blocked by the caspase inhibitor Z-VAD-fmk and partially blocked by Ac-DEVD-fmk, suggesting that SN50-mediated apoptosis is caspase-dependent. Interestingly, apoptosis induced by NF-kappa B suppression, in contrast to that induced by TPEN (N,N,N',N'-tetrakis [2-pyridylmethyl]ethylenediamine) or soluble Fas ligand (CD95), was observed in the absence of active death effector proteases caspase-1-like (IL-1 converting enzyme), caspase-3-like (CPP32/Yama/apopain), and caspase-6-like and without cleavage of caspase-3 substrates poly(ADP-ribose) polymerase and DNA fragmentation factor-45. These findings suggest either low level of activation is required or that different caspases are involved. Preactivation of T cells resulting in NF-kappa B nuclear translocation protected cells from SN50-induced apoptosis. Our findings demonstrate an essential role of NF-kappa B in survival of naive PBL.

Mechanisms of apoptosis in T cells from patients with renal cell carcinoma.

Tumors may escape immune recognition and destruction through the induction of apoptosis in activated T lymphocytes. Results from several laboratories suggest that FasL (L/CD95L) expression in tumors may be responsible for this process. In this study of patients with renal cell carcinoma (RCC), we provide evidence for two mechanisms of T-cell apoptosis. One mechanism involves the induction of apoptosis via FasL expression in tumor cells. This is supported by several observations, including the fact that tumor cells in situ as well as cultured cell lines expressed FasL mRNA and protein by a variety of techniques. The FasL in RCC is functional because in coculture experiments, FasL+ tumors induced apoptosis in Fas-sensitive Jurkat T cells and in activated peripheral blood T cells but not in resting peripheral blood T cells. Most importantly, antibody to FasL partially blocked apoptosis of the activated T cells. Moreover, Fas was expressed by T cells derived from the peripheral blood (53% median) and tumor (44.3% median) of RCC patients. Finally, in situ staining for DNA breaks demonstrated apoptosis in a subset of T cells infiltrating renal tumors. These studies also identified a second mechanism of apoptosis in RCC patient peripheral T cells. Whereas these cells did not display DNA breaks when freshly isolated or after culture for 24 h in medium, peripheral blood T cells from RCC patients underwent activation-induced cell death after stimulation with either phorbol 12-myristate 13-acetate/ionomycin or anti-CD3/CD28 antibodies. Apoptosis mediated by exposure to FasL in tumor cells or through T-cell activation may contribute to the failure of RCC patients to develop an effective T-cell-mediated antitumor response.

Signal transduction abnormalities in T lymphocytes from patients with advanced renal carcinoma: clinical relevance and effects of cytokine therapy.

Studies have demonstrated abnormalities of the CD3/T-cell antigen receptor (TCR) and pathways of signal transduction in T lymphocytes from animals and patients with advanced malignancy. Diminished expression of TCRzeta and p56(lck) that are associated with the TCR and reduced nuclear localization of RelA containing nuclear factor kappaB (NFkappaB) complexes have been noted. These defects have been described in T cells from patients with malignant melanoma, renal cell carcinoma (RCC), ovarian cancer, and colorectal cancer. Preliminary observations also indicate possible correlation with clinical variables such as stage in selected instances. To further characterize altered expression of TCRzeta, p56(lck), and impaired activation of NFkappaB, T lymphocytes were obtained from 65 patients with RCC, the majority of whom were receiving combination cytokine therapy [interleukin (IL)-2, IFN alpha-containing regimens] and 37 control individuals. In 29 of these patients, levels of TCRzeta and p56(lck) were determined by Western blots of T-cell lysates and semiquantitated using densitometry. Relative levels were then correlated with a series of clinical variables including response to therapy, performance status, survival, disease sites, age, and others. In another group of 28 patients (three individuals from the first group), the frequency of abnormal NFkappaB activation was studied using electrophoretic mobility shift assays after activation of T cells with phorbol myristate acetate/ionomycin or anti-CD3 monoclonal antibody. Changes in these signaling molecules during cytokine treatment were also investigated. TCRzeta and p56(lck) were detected in the peripheral blood T cells in 27 of 29 patients, and overall, reduced levels were noted visually in 12 of 29 (41%) and 13 of 29 (45%) individuals, respectively. When levels were semiquantitated using densitometry, significant decreases of TCRzeta (P = 0.029) and p56(lck) (P = 0.029) but not CD3epsilon (P = 0.131), compared with control levels, were found. In patients treated with IL-2/IFN alpha-based therapy, relative levels of TCRzeta increased significantly (P = 0.002) on day 15 of cycle one compared with the baseline. Correlations of TCRzeta or p56(lck) levels with response or disease variables, except for lower TCRzeta levels (P < 0.001) in the presence of bone metastases, were not found. Abnormal NFkappaB activation after stimulation with phorbol myristate acetate/ionomycin and/or anti-CD3 monoclonal antibody was found in 59% of patients (17 of 28) and was not accounted for by the advanced age of the study cohort. Activation of NFkappaB in peripheral blood T cells was inducible during cytokine therapy in four of six individuals who displayed impaired NFkappaB activity prior to therapy. Moreover, impaired activation of NFkappaB does not appear linked to a reduction of TCRzeta expression, because in five patients, normal TCRzeta levels were present although kappaB binding was not inducible. In the majority of patients with advanced RCC, peripheral blood T cells express TCRzeta and p56(lck), and in a subset, reduced levels of these TCRzeta associated molecules are seen that may increase during cytokine-based therapy. Abnormal activation of NFkappaB is also present in >50% of patients and may also revert to normal during IL-2/IFN alpha-based treatment. This alteration in NFkappaB activation occurred in the presence of normal expression of TCRzeta-associated signaling elements. The clinical significance of these findings remains unclear.

Mortality of industrial workers exposed to acrylonitrile.

OBJECTIVES: This study was designed to evaluate the relationship between occupational exposure to acrylonitrile and cancer mortality. MATERIALS AND METHODS: Workers (18079 white men, 4293 white women, 2191 nonwhite men, and 897 nonwhite women) employed in acrylonitrile production or use in the 1950s through 1983 were followed through 1989 for vital status and cause of death. Exposure-response relationships were evaluated from quantitative estimates of historical exposures. Tobacco use was determined for a sample of workers to assess potential confounding. Mortality rates between the exposed and unexposed workers in the cohort were compared using the Poisson regression. RESULTS: Analyses by cumulative, average, peak, intensity, duration, and lagged exposure revealed no elevated risk of cancers of the stomach, brain, breast, prostate or lymphatic and hematopoietic systems. Mortality from lung cancer was elevated for the highest quintile of cumulative exposure. When the decile categories were used, the relative risk did not continue to increase at higher levels. Adjustment for cigarette use reduced the risk for lung cancer only slightly. Separate analyses for wage and salaried workers, long-term and short-term workers, fiber and nonfiber plants, and individual plants revealed no clear exposure-response patterns. CONCLUSIONS: The results indicate that exposure to acrylonitrile at the levels studied is not associated with an increased relative risk for most cancers of a priori interest. The excess of lung cancer in the highest quintile of cumulative exposure may indicate carcinogenic activity at the highest levels of exposure, but analyses of exposure-response do not provide strong or consistent evidence for a causal association.

Exposure assessment for a study of workers exposed to acrylonitrile.

Procedures used to develop estimates of exposure to acrylonitrile for a cohort study (>25000 workers in 8 monomer, fiber, and resin companies from 1952 to 1983) are presented. Visits to the companies were made, interviews of workers were conducted, historical records were made, and measurements were taken. On the basis of similar tasks, locations, other exposures, and a similar distribution of exposures to acrylonitrile, 3600 exposure groups were formed. Special procedures were used to reduce the misclassification of workers performing tasks that varied in time but that were inadequately reflected in the job title. A software program organized and retained all exposure information on each exposure group. Quantitative estimates of acrylonitrile exposure were developed using a hierarchical approach in a software program that documented the derivation of each estimate and facilitated data review. Two of the estimation methods were evaluated in a comparison with measurement data.

Identification and tissue-specific expression of PDE7 phosphodiesterase splice variants.

Type 7 cyclic nucleotide phosphodiesterases (PDE7s) are a newly described family of enzymes having high affinity and specificity for cAMP. However, little is known about their structure, function, or regulation. We have isolated a mouse skeletal muscle cDNA representing a new alternative splice variant (PDE7A2) of the PDE7 gene. The ORF encodes a 456-amino acid protein having a predicted molecular weight of 52.4 kDa. The 5' end of the mouse PDE7A2 is divergent from the 5' end of the human PDE7A1 sequence and is more hydrophobic. A comparison of the 5' ends of the two cDNA clones with human genomic sequence indicates that they represent alternate splice products rather than species variation. RNase protection analysis of several mouse tissues indicates that PDE7 is expressed widely with highest levels in skeletal muscle. HPLC fractionation and Western blot analysis of two human lymphocyte T-cell lines shows that an unknown PDE activity described by Ichimura and Kase [Ichimura, M. & Kase, H. (1993) Biochem. Biophys. Res. Commun. 193, 985-990] is most likely to be PDE7A1. A single immunoreactive band of approximately 55 kDa, which comigrates with PDE7A1, is seen in fractions of the HPLC profile containing this activity suggesting that the original human PDE7A1 clone contains a full-length ORF, and is not truncated at the 5' end as was originally postulated. In a human lymphocyte B-cell line and also in mouse skeletal muscle, a large amount of PDE7 mRNA but little PDE7 protein or activity is expressed suggesting that the translation or stability of PDE7 protein may be highly regulated in these tissues.

Position of the wrist associated with the lowest carpal-tunnel pressure: implications for splint design.

Increased carpal-tunnel pressure has been implicated in the pathophysiology of carpal tunnel syndrome, but it is not known whether splints that immobilize the wrist in a functional position of extension minimize carpal tunnel pressure. To determine the position of the wrist that results in the lowest carpal-tunnel pressure, twenty control subjects and four patients who had carpal tunnel syndrome were evaluated with use of a new, dynamic method that continuously measures carpal tunnel pressure throughout the range of motion of the wrist. The pressure was measured by means of a pressure transducer connected to a flexible catheter that had been inserted into the carpal canal. The position of the wrist was measured simultaneously with use of a two-axis electrogoniometer. Aided by a computer monitor that displayed a moving line of real-time carpal-tunnel pressure, each subject was instructed to move the wrist throughout the range of motion and to adjust it to the position that corresponded to the lowest carpal-tunnel pressure. For the control subjects, the lowest carpal-tunnel pressure averaged 8 +/- 4 millimeters of mercury (1.07 +/- 0.53 kilopascals), and the average position of the wrist associated with the lowest pressure was 2 +/- 9 degrees of extension and 2 +/- 6 degrees of ulnar deviation.(ABSTRACT TRUNCATED AT 250 WORDS)