Paradoxical worsening of chest radiographs secondary to immune reconstitution syndrome (IRIS) in a patient with advanced HIV infection and Rhodococcus pneumonia.

We present a rare case of post-antiretroviral therapy (ART) paradoxically worsening of radiological findings in a patient with advanced HIV-infection on treatment for Rhodococcus pneumonia who was misdiagnosed with pulmonary tuberculosis. Despite clinical improvement, serial chest radiographs showed deteriorations a month after starting ART. This was attributed to Immune Reconstitution Inflammatory Syndrome (IRIS) which spontaneously resolved without any treatment.

Procalcitonin and C-reactive protein as markers of infection in systemic lupus erythematosus: the controversy continues.

OBJECTIVE: The objective of this paper is to investigate the utility of serum procalcitonin (PCT) and C-reactive protein (CRP) as markers of infection in systemic lupus erythematosus (SLE) patients. PATIENTS AND METHODS: Sixty-nine SLE patients with symptoms and signs of infection proved by culture and/or a favorable response to antibiotics and 69 SLE patients without infection were included. Serum PCT and plasma high-sensitivity CRP were assessed by an enzyme-linked immunosorbent assay. RESULTS: SLE patients with infection had a significantly higher level of CRP than those without infection ((median (IQR) 104.5 (25.5-100.9) and 10.3 (5.4-23.1) mg/l, respectively), p<0.001). CONCLUSION: Serum PCT could not differentiate SLE patients with or without bacterial infection in this study, while the utility of CRP as a marker of infection has been confirmed.

Seroprevalence of Helicobacter pylori Infection in Renal Transplant Recipient Attending Duhok Kidney Disease Center.

INTRODUCTION: Helicobacter pylori plays an important role in the development of gastritis, peptic ulcer disease, and gastric cancer in the general population. Although dyspeptic complaints are frequent in renal transplant subjects, the role of H pylori in such complaints is not clear. This study was conducted to examine the prevalence of H pylori infection in patients with renal transplant and its role in the development of upper gastrointestinal tract (GIT) symptoms. MATERIALS AND METHODS: We compared the prevalence of H pylori in 109 renal transplant subjects with the prevalence of 161 healthy volunteers using an enzyme-linked immunosorbent assay to measure anti-H pylori IgG. Then, within the transplant group, we stratified our data in accordance with different variables, including upper GIT symptoms, sex, education level, smoking, and dialysis history. RESULTS: The prevalence of H pylori in the control group was 111 of 161 (68.9%), which was significantly higher than that found in renal transplant subjects (48/109; 43.04%; P = .001). Within the transplant group, no association was found between immunoglobulin G positivity and different variables including upper GIT symptoms, sex, education level, smoking, and dialysis history. CONCLUSIONS: The prevalence of H pylori was lower in transplant subjects than that of general population. Upper GIT symptoms were equally found in both groups. Hence, the routine eradication of H pylori might not be indicated.

Heterodimeric l-amino acid oxidase enzymes from Egyptian Cerastes cerastes venom: Purification, biochemical characterization and partial amino acid sequencing.

Two l-amino acid oxidase enzyme isoforms, Cc-LAAOI and Cc-LAAOII were purified to apparent homogeneity from Cerastes cerastes venom in a sequential two-step chromatographic protocol including; gel filtration and anion exchange chromatography. The native molecular weights of the isoforms were 115 kDa as determined by gel filtration on calibrated Sephacryl S-200 column, while the monomeric molecular weights of the enzymes were, 60, 56 kDa and 60, 53 kDa for LAAOI and LAAOII, respectively. The tryptic peptides of the two isoforms share high sequence homology with other snake venom l-amino acid oxidases. The optimal pH and temperature values of Cc-LAAOI and Cc-LAAOII were 7.8, 50 °C and 7, 60 °C, respectively. The two isoenzymes were thermally stable up to 70 °C. The K m and V max values were 0.67 mM, 0.135 µmol/min for LAAOI and 0.82 mM, 0.087 µmol/min for LAAOII. Both isoenzymes displayed high catalytic preference to long-chain, hydrophobic and aromatic amino acids. The Mn2 + ion markedly increased the LAAO activity for both purified isoforms, while Na+, K+, Ca2 +, Mg2 + and Ba2 + ions showed a non-significant increase in the enzymatic activity of both isoforms. Furthermore, Zn2 +, Ni2 +, Co2 +, Cu2 + and AL3 + ions markedly inhibited the LAAOI and LAAOII activities. l-Cysteine and reduced glutathione completely inhibited the LAAO activity of both isoenzymes, whereas, ß-mercaptoethanol, O-phenanthroline and PMSF completely inhibited the enzymatic activity of LAAOII. Furthermore, iodoacitic acid inhibited the enzymatic activity of LAAOII by 46% and had no effect on the LAAOI activity.

Association of HLA locus variant in Parkinson's disease.

A variant (rs3129882) in the genome-wide association study (GWAS)-linked variant [in the human leukocyte antigen (HLA) gene region] has been reported to associate with an increased risk of Parkinson's disease (PD) in Caucasian population. Studies among Chinese are limited. To address this, we analysed rs3129882 in a total of 1312 subjects of Chinese ethnicity from independent Asian centers comprising of 675 controls and 637 PD cases. The rs3129882 variant was associated with a decreased risk in our ethnic Chinese PD patients. Logistic regression analysis taking into consideration variables of age, gender and race showed that allele A reduced the risk of PD via a dominant model [odds ratio (OR) = 0.77, 95% confidence interval (CI) = 0.62, 0.96, p = 0.018]. As HLA is a highly polymorphic region, it is possible that ethnic-specific effect or environmental agents may modulate the effect of this GWAS-linked locus in influencing the risk of PD.

The prognosis of fixed dystonia: a follow-up study.

BACKGROUND: The syndrome of fixed dystonia includes both CRPS-dystonia and psychogenic dystonia. The underlying mechanisms are unclear, but a high prevalence of neuropsychiatric illness has previously been reported. METHODS: Clinical and neuropsychiatric follow-up study by telephone and self-administered instruments (HADS, SDQ-20, DES II, EQ-5D), on 41 patients with fixed dystonia after a mean of 7.6 (+/-3.6) years. RESULTS: We obtained information on clinical outcome in 35 (85.4%) patients and neuropsychiatric questionnaire data in 22 (53.7%). Eighty-three percent were women. Thirty-one percent had worsened, 46% were the same and 23% had improved, of whom 6% had major remissions. At follow-up, mean duration of illness was 11.8 (+/-4.9) years and mean age 43.2 (+/-14.8) years. Except for 1 patient who was re-diagnosed with corticobasal degeneration, the diagnosis remained unchanged in others. Forty-one percent had scores indicating anxiety and 18% indicating depression; 18% scored within the range of dissociative/somatoform disorders on DES II and 19% on SDQ-20. The mean EQ-5D index and VAS scores were 0.34 and 56.1%. Comparison between the 3 outcome groups revealed significant difference only in the EQ-5D (p=0.003). Only baseline CRPS predicted a worse outcome (chi(2)=0.006). CONCLUSIONS: Our findings revealed that the prognosis of this syndrome is poor, with improvement in less than 25% of patients, major remission in only 6% and continued worsening in a third. A high rate of neuropsychiatric findings was noted and new neuropsychiatric features had occurred in some. Average health status was poor. Of the baseline parameters, only CRPS predicted poorer outcome.

Assessment of the anti-Naja haje antibodies elicited in a low dose multi-site immunization protocol.

The horse antibodies to Naja haje (Nh) elicited in a low dose multi-site immunization protocol were investigated from binding perspective in the context of antivenom maturation. We found that, this protocol evoked the production of lethality neutralizing avid antibodies in the first round of immunization which increases over the successive immunization rounds. The changes and the relative changes in the antibody parameters of each horse were taken as a measure for the efficacy of its immune system to respond by gaining and magnifying the antivenom parameters. While the avidity increases over the successive immunization rounds towards a maturation ceiling, the avidity gain and relative gain decreases. While the ED(50) gain increases over the immunization rounds, the relative ED(50) gain of the strong responder horse-1 is steadier than for the weak responders horse-2 and horse-3. The profiles for the gain and the relative gain of ELISA titers by three horses are pronouncedly different, reflecting great variations in the immune response by the individual animals. A late antigen dependent avidity pulse was detected after the last dose of the second immunization period, demonstrating that antibody maturation continues during the rest periods. The avidity pulse appears as an increase in avidity which returns to a base level within four weeks. These results indicted that maturation of antibodies to lethality neutralization relevant venom epitopes rather than maturation of antivenoms is the most important potency determining factor. Eventually, a main advantage of the low dose multi-site immunization protocol is the production of highly avid antibodies at the primary immunizations which is mostly crucial for the production of highly potent antivenoms.

Avidity based discrimination of venoms from two Egyptian Echis species.

Species-specific antibodies (Abs) to venoms from two species of the genus Echis were prepared by adsorption of monovalent and affinity purified anti-Echis Abs to the heterologous venom matrices (VMs). The avidity of the Abs to the homologous and the heterologous venoms is constantly ranked in the order; monovalent Ab>cross-reactive Ab>species-specific Ab. The avidity of the species-specific Abs to the homologous venoms of Echis coloratus (Eco) and Echis carinatus pyramidum (Epy) were 1.20+/-0.26 and 1.10+/-0.29, respectively. The avidity of the species-specific Abs to the heterologous venoms was too low to be detected. These results demonstrate for the first time that the avidity can be used to discriminate the immunologically high cross-reactive venoms. The discrimination of (14/14) venom samples of the two species on avidity bases confirmed the reliability and the specificity of the assay.

Effect of scrotal insulation on clusterin-positive cells in ram semen and their relationship to semen quality.

Scrotal contents of 2 rams were insulated for 96 hours and the fraction (as a percentage) of clusterin-positive cells (CPCs) and its relationship to semen quality was investigated. Semen collection was started 18 days before insulation and was terminated on day 78 and day 63 after insulation in animals 1 and 2, respectively. Sperm clusterin was localized by immunostaining with anti-bovine clusterin antibody (anti-bCAb) and fluorescein isothiocyanate-conjugated immunoglobulin G. Scrotal insulation led to deterioration of semen quality and increased the percentage of CPCs in both rams. Two types of sperm reactivity were observed: an extensive, intensive staining pattern (ESP); and a localized, less-intensive staining pattern (LSP). The percentage of ESP-CPCs began to increase from day 6 and reached 88.8% and 100% on day 15 after insulation in animals 1 and 2, respectively. The increase in CPCs coincided with the presence of a high percentage of teratoid forms (88.3%) in semen from animal 1, and detached heads (81.4%) in semen from animal 2. After normal semen production was restored on day 60 in animal 1, the percentages of ESP-CPCs and LSP-CPCs returned to preinsulation rates, whereas only the ESP-CPCs returned to normal in animal 2. A negative relationship was observed between ESP-CPCs and total sperm/ejaculate (r = -.62), motility (r = -.78), viability (r = -.68), and filtration rate (r = -.71) in semen from animal 1. Conversely, a positive relationship was seen between ESP-CPCs and total abnormal spermatozoa (r = .82). Similar results were obtained in semen from animal 2. CPCs were nearly completely absent in glass wool-Sephadex (GWS)-filtered semen, suggesting a role for clusterin in the process of trapping abnormal spermatozoa in the GWS filters. We conclude that aberrant spermatogenesis induced by scrotal insulation increases the percentage of CPCs in ram semen. We suggest that the percentage of CPCs in ram semen could be a useful marker in poor-quality ejaculates.

Localization of clusterin on freeze-preserved bull spermatozoa before and after glass wool-sephadex filtration.

Clusterin is a major protein in bull reproductive tract secretions and sperm membrane extract. A polyclonal antibody was produced against clusterin from bull cauda epididymal fluid (CEF) and used for the localization of the protein on bull spermatozoa. Immunoblotting of unreduced bovine samples showed that the anticlusterin antibody reacted with a protein of approximately 94- to 100-kd in rete testis fluid (RTF), a approximately 57- to 76-kd protein in CEF, and with a approximately 57- to 60-kd protein from cauda epididymal sperm membrane extract. The antibody also reacted with stallion RTF and both ram CEF and RTF at relative molecular weights (Mr) that were consistent with the anticipated size of clusterin in these species. Less intense immunostaining was observed for a protein of about 2 times the predicted size of clusterin in unreduced ovine RTF, suggesting the presence of multimers of clusterin in ovine RTF. Also, a dimeric clusterin-sized protein was detected in reduced bovine CEF, suggesting the presence of unprocessed clusterin in bovine CEF. By immunofluorescence, clusterin was detected on only a small fraction of bull spermatozoa, which were morphologically abnormal. Neither permeabilization nor the method of dilution affected the reactivity of the antibody with spermatozoa (P > .05). Average clusterin-positive spermatozoa (CPS) in unpermeabilized, permeabilized, abruptly diluted, and gradually diluted semen were 10.1%, 11.3%, 15.0%, and 14.4%, respectively. CPS were eliminated from semen after filtration through glass wool-Sephadex (GWS) columns. Average CPS in unfiltered and filtered semen were 14.3% and 1.1%, respectively. We conclude that sperm clusterin in bull semen is associated with morphologically abnormal spermatozoa and that clusterin is implicated in the process of abnormal spermatozoa trapping in GWS columns. We suggest that the fraction of CPS in bull semen is a potential marker for poor semen quality.

Epididymal specificity and androgen regulation of rat EP2.

In primates, expression of the EP2 gene is androgen-dependent and epididymis-specific. EP2 mRNA expression was investigated in caput, corpus, and cauda regions of rat epididymis and in 15 other rat tissues. Polymerase chain reaction and Northern analyses showed that rat EP2 is expressed predominantly in the proximal caput epididymidis. EP2 mRNA expression was determined in proximal epididymides from castrated, sham-operated, and efferent duct-ligated rats. In castrated rats, EP2 mRNA decreased to <10% of that in sham-operated rats between Days 3 and 4 postcastration, demonstrating the androgen dependence of EP2 expression. In epididymides ligated unilaterally at the efferent ducts, EP2 mRNA levels were approximately equal to those in the unligated contralateral epididymides or in sham-operated rats, indicating that EP2 expression does not depend on testicular factors. In bilaterally castrated rats, immediate and delayed testosterone replacement showed the dependence of EP2 expression on circulating androgens. Injection of testosterone propionate (TP) on Days 0, 1, 2, and 3 postcastration maintained EP2 mRNA levels approximately equal to those in sham-operated rats. Starting at Day 4 postcastration, daily injection of TP for 7 days restored EP2 mRNA to approximately normal levels. These data indicate for the rat that EP2 is expressed specifically in the proximal caput epididymidis and that its expression depends on circulating androgens but not on testicular factors.

Correlation between clusterin-positive spermatozoa determined by flow cytometry in bull semen and fertility.

The objectives were to 1) develop a rapid and accurate method for detection of clusterin-positive spermatozoa (CPS) in bull semen and 2) determine the utility of incidence of CPS for prediction of fertility of bull semen in comparison to routine semen quality traits. Semen from 3 bulls was immunostained with anti-bovine clusterin antibody and with FITC-conjugated anti-rabbit IgG for method development. Clusterin-positive spermatozoa were determined by flow cytometry (FCM) and fluorescence microscopy, and results were compared by paired t test. There was no difference between FCM and microscopic techniques (P = .81). Flow cytometry was then used for determination of CPS in semen of 48 bulls with known fertility. Significant inverse relationships were found between the percentage of CPS and raw nonreturn rate (r = -.30), adjusted nonreturn rate (r = -.58), and estimated relative conception rate (ERCR; r = -.60). Estimated relative conception rate is potentially a very accurate method for determining fertility, and it resulted in highest correlation with CPS. An inverse relationship was observed between the percentage of CPS and prefreeze and postfreeze motility (r = -.51), whereas a direct relationship was found between CPS and primary, secondary, tertiary, and total sperm abnormalities (r = .52, .77, .32, and .58, respectively). The fractions of motile and abnormal spermatozoa, with the exception of tertiary abnormalities, were inversely correlated with 2 or more of the fertility estimates, but none of them showed the characteristic increase in correlation with improvement of accuracy of fertility estimate as demonstrated by CPS. We conclude that FCM is useful for objective and efficient detection of CPS in bull semen. The results suggest that the percentage of CPS in bull semen is potentially a better predictor of fertility than sperm motility or abnormal morphology.

Pitfall of an internal control plasmid: response of Renilla luciferase (pRL-TK) plasmid to dihydrotestosterone and dexamethasone.

The thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK) is commonly used as a control for transfection efficiency in the Dual-Luciferase Reporter Assay System. While investigating hormone response elements in the promoters of the androgen-dependent, epididymis-specific EP2 gene, we found that hormone treatment affected the luciferase activity of pRL-TK-transfected cells. In African Green Monkey Kidney (CV-1) cells, cotransfected transiently with a hormone-responsive promoter-firefly luciferase reporter plasmid and with pRL-TK, Renilla luciferase activity increased in response to dihydrotestosterone (DHT) and decreased in response to dexamethasone (DEX). When a thromboxane synthase promoter Renilla luciferase plasmid (pRL-TS) was used in place of pRL-TK, Renilla luciferase activity remained constant in CV-1 cells treated with DHT but decreased in CV-1 cells treated with DEX. In transfection studies, internal control plasmid expression in response to treatment must be carefully monitored to ensure proper interpretation of normalized results.

Reproductive tract secretions and bull spermatozoa contain different clusterin isoforms that cluster cells and inhibit complement-induced cytolysis.

Clusterin from bull rete testis fluid (RTF), cauda epididymal fluid (CEF), and octyl-beta-D-glucopyranoside extract of cauda epididymal sperm (CES) was identified and characterized using monoclonal and polyclonal antibodies (Abs) developed against ram clusterin and a beta-subunit-specific oligopeptide of porcine clusterin. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting showed that bovine RTF clusterin had dimeric and monomeric molecular weights (M(r)s) of approximately 94 kDa and of 42 and 43 kDa, respectively. Clusterin in CEF and CES had similar dimeric M(r)s (74 kDa). Reduced CEF clusterin appeared as three monomers (M(r)=40, 39, and 38 kDa), whereas reduced CES clusterin appeared only at M(r)40 kDa. Enzymatic deglycosylation resulted in similar M(r)s of clusterin from RTF, CEF, and CES. The M(r) of RTF clusterin decreased from 94 kDa to 51 kDa, indicating a carbohydrate content of 45%. After deglycosylation, the M(r) of the CEF clusterin decreased from 74 kDa to two distinct bands at 51 and 50 kDa (with carbohydrate contents of 31 and 32%, respectively), suggesting that two isoforms of the heterodimeric protein are present because of the two isoforms of the alpha-subunit. Under nonreduced conditions, a beta-subunit-specific Ab reacted with M(r) of 36-38 kDa, indicating the existence of free clusterin beta-subunits in CES. RTF, CEF, and CES extracts all caused mouse fibroblastic L-cell aggregation. CEF cell aggregation was inhibited by Hyb-17 Ab but not by other Abs. Both RTF and CEF caused a dose-dependent inhibition of complement-induced cytolysis, although RTF clusterin was more potent than CEF clusterin. We conclude that several isoforms of clusterin occur in the bull reproductive tract and that the variation in carbohydrate content among these isoforms may affect the biological or functional activity of the protein.

Apoptosis in chronic gastritis: evaluation of the gastric mucosa by DNA flow cytometry and the expression of the high molecular weight cytokeratin.

BACKGROUND/AIMS: A series of premalignant lesions, including chronic gastritis (CG), intestinal metaplasia (IM) and dysplasia are associated with gastric carcinogenesis. The present study aimed to define these precancerous gastric lesions further by the study of the cellular DNA using flow cytometry, and the expression of the high molecular weight (68 KDa) Cytokeratin "CK1" proposed as a marker for epithelial cells dying by apoptosis. MATERIAL AND METHODS: Multiple antral biopsies from each of 92 cases with gastric dyspepsia were subjected for DNA content analysis using flow cytometry, and immunostaining using anti-CK1 monoclonal antibody. RESULTS: Chronic gastritis (CG) was present in 85 (92.4%) of cases, 14/85 (16.5%) cases showed chronic superficial gastritis (CSG), and 71/85 (83.5%) cases were chronic atrophic gastritis (CAG). Sixty two of the 85 (74.7%) cases with CG revealed variable degrees of activities. A hypodiploid "Sub-G1" peak was detected in 35 of 85 cases with CG. This peak was significantly higher in active chronic gastritis (ACG) than in the inactive (ICG) cases (p < 0.005). Proliferative activity of cases with CG was higher than in normal cases (p < 0.05) and in cases with ACG than in ICG (p < 0.05). Abnormal DNA-content (aneuploidy) was present in 16 (18.8%) of the 85 cases with CG. The presence of gastric epithelial cells with morphological changes typical of apoptosis in cases showing hypodiploid "Sub-G1" peak, high proliferation, and DNA-aneuploidy, suggests that these cells may be apoptotic bodies. Mild degree of apoptosis was present in some cases (57%) with histologically normal mucosa, while dense apoptotic bodies occurred in 87% of cases with chronic gastritis. These apoptotic bodies were constantly expressing CK1, except those in normal mucosa, suggesting that CK1 can be used as a marker for dying epithelial cells by apoptosis. CK1 was detected in 16 (100%) aneuploid cases which also showed apoptosis. CONCLUSION: The presence of apoptotic bodies in cases with chronic gastritis especially in those showing DNA-aneuploidy, may accounts for the deletion of cells with altered DNA.

Adult dengue haemorrhagic fever at Kuala Lumpur Hospital: retrospective study of 102 cases.

A retrospective study involving 102 adults with dengue haemorrhagic fever (DHF) was conducted to investigate the demographic aspect, clinical presenting features, laboratory investigations, complications, and mortality associated with the disease. The clinical diagnosis of DHF was in accordance with WHO recommendations. Epistaxis, gingivitis, haematemesis and gastritis were among the common complications. Platelet levels tended to decline from a higher value on admission (mean 67,000/mm3) to lower levels on subsequent days, with the lowest (mean 61,000/mm3) being on day 6 of the fever. Hyponatraemia (46.8%) was commonly observed. Morbidity of DHF was significant (29.4%) but the case fatality rate remained low (2.0%) in our adults, suggesting that adults are less likely than children to suffer from shock syndrome.

Heavy metal content (Hg2+, Cd2+, Pb2+) in various body parts: its impact on cholinesterase activity and binding glycoproteins in the grasshopper Aiolopus thalassinus adults.

Various toxicological symptoms were observed in Aiolopus thalassinus (Fabr.) adults which resulted from either contaminated soil or from being fed HgCl2-, CdCl2-, and PbCl2-treated diet. Many insects had abnormal wings which were present until the F3 untreated generation. Heavy metals were stored in different body parts, most of Cd2+ and Hg2+ were found in the testes, followed by the gut. Lead was enriched in all organs and body parts of the treated grasshoppers compared to the control. The highest lead concentrations were found in testes, wings, gut, and ovaries. In the treated generation ChE activity was reduced to about 23% compared to the untreated grasshoppers. In contrast to this, lead increased ChE activity to about 14%. In the following F2 untreated adults, ChE activity was normal; no long-term effect was found. In the supernatant of treated adults a Cd-type glycoprotein was found which can bind Cd ions; its MW was determined by electrophoresis and reference proteins at about 14,450 Da.

Long-term effects of heavy metals in food on developmental stages of Aiolopus thalassinus (Saltatoria: Acrididae).

Newly hatched F1 nymphs of Aiolopus thalassinus (Fabr.) were fed on food treated with various concentrations of HgCl2, CdCl2, and PbCl2 until the end of adult life. Toxicological observations were followed in the F1 generation and in the F2 generation derived from the heavy metal-loaded F1 parents. The highest concentration of the heavy metal caused 100% mortality of the F1 adults within four weeks. The nymphal duration of the F1 and F2 generations was significantly prolonged after Hg and Cd exposure, but the F1 of the group treated with lead was not affected. The fresh body weight of adults was significantly reduced in the F1 generation of most treatments and in the resulting untreated F2. The lifespan of the F1 adults was shortened. In the F2 generation, although the lifespan was somewhat longer, generally it was still shorter than that of the control adults. The mean egg number laid by F1 adults fed on food contaminated with Hg or Cd was decreased. This decrease was more pronounced in the case of Cd than Hg. In the females fed on food treated with Pb the reduction of the number of egg pods was not remarkable. The hatchability of the eggs laid by F1 females was significantly reduced as compared to the control. The viability of the eggs laid by F2 adults was somewhat decreased due to either a reduced number of egg pods or to a lower rate of hatchability, especially in the case of Cd. The treated adults frequently displayed weakness in their legs, difficulties in walking, tremors, and nervous movements.(ABSTRACT TRUNCATED AT 250 WORDS)