Comparison of Microbial Contamination Levels Among Hospital Operating Rooms and Industrial Clean Rooms

Microbial contamination in industrial clean rooms was compared quantitatively and qualitatively with that of hospital operating rooms. The number of aerobic mesophilic microorganisms which accumulated on stainless-steel strips exposed for periods up to 21 weeks to the intramural air of four operating rooms was at least 1 log higher than the accumulation on strips exposed in four clean rooms, and was essentially the same as that found in two factory areas. Volumetric air samplings showed that there were significantly higher numbers of airborne viable particles per cubic foot of air in operating rooms than in industrial clean rooms. In contrast to clean rooms, where most of the airborne contaminants were those associated with human hair, skin, and respiratory tract, the hospital operating rooms showed a very high level of microorganisms associated with dust and soil.     

Evaluation of dust respirators for elimination of mouse aeroallergens

The efficiency of various dust respirators for eliminating mouse allergens [mouse urine proteins (MUP), pelts proteins (MPP) and serum albumin (MSA)] were evaluated with use of low-volume air samplers and immunochemical methods. Three kinds of dust respirators from one manufacturer which have different efficacy in the exclusion of dust particles were put on the fiber glass filter in each air sampler. Then the air in a mouse housing room was sampled. The allergens passed through the respirators, were trapped in the fiber glass filters, and then extracted from the filters. The allergens of MUP and MPP in the extract were measured by an inhibition method of fluorometric enzyme-linked immunosorbent assay (ELISA) for IgE antibody and those of MSA measured by a fluorometric sandwich ELISA. The respirator with the lowest capability of exclusion was found to eliminate 65-86% of respective allergens. The other two respirators with higher powers eliminated 98% of MUP. MPP and MSA were eliminated to undetectable levels through these respirators. This study provided a means for the evaluation of dust respirators for animal aeroallergens.     

Trichothecene mycotoxins in aerosolized conidia of Stachybotrys atra

Stachybotrys atra is the etiologic agent of stachybotryotoxicosis, and this fungus and its trichothecene mycotoxins were recently implicated in an outbreak of unexplained illness in homes. S. atra was grown on sterile rice, autoclaved, dried, and then aerosolized by acoustic vibration. The distribution of particles (mass and number) was monitored on an aerodynamic particle sizer interfaced with a computer. Dust was collected on preweighed glass-fiber filters and extracted with 90% aqueous methanol. Extracts were tested for the ability to inhibit protein synthesis in rat alveolar macrophages, the ability to inhibit the proliferation of mouse thymocytes, and the presence of specific trichothecene mycotoxins. Virtually all of the particles were less than 15 micron in aerodynamic diameter, and the mass median diameter was 5 micron. Thus, most of the particles were respirable. Microscopic analysis of the generated dust revealed that ca. 85% of the dust particles were conidia of S. atra, another 6% were hyphal fragments, and the remainder of the particles were unidentifiable. Thus, greater than 90% of the particles were of fungal origin. The extracts strongly inhibited protein synthesis and thymocyte proliferation. Purified satratoxin H was also highly toxic in the same systems. Each of the individual filters contained satratoxin H (average, 9.5 ng/mg of dust). Satratoxin G and trichoverrols A and B were found in lesser amounts in some, but not all, of the filters. The limit of analysis is ca. 50 ng. These results establish that the conidia of S. atra contain trichothecene mycotoxins. In view of the potent toxicity of the trichothecenes, the inhalation of aerosols containing high concentrations of these conidia could be a potential hazard to health.     

Observation on the air-borne bacteria and ammonia (NS3) gas in laboratory animal facility with rotary heat exchanger]

The number of air-borne bacteria in air ducts and barrierred laboratory animal rooms with the so-called econovent rotary heat exchanger, were checked monthly during a year by the pin-hole sumpler method for air ducts and Koch method for animal rooms. Also, concentration of ammonia was checked with the same samples by gas impinger. No significantly difference in number of air-borne bacteria was seen between before and after passing the econovent. Those passing through HEPA filter was not detected. There were more air-borne bacteria in animal rooms, outside locker room and shower room than in the corridor, utensil storage, inside locker room and pass box. No ammonia were detected in the outdoor, but exhaust air duct after passing the econovent contained very small amount of ammonia. On the other hand, high concentration of ammonia were preserved in the supplying air duct, exhaust air duct and mice and rats rooms, about 86% of ammonia in exhaust air duct returned back into the supplying air duct. No influences on reproduction in mice and rats were recognized.     

Trichothecene mycotoxins in aerosolized conidia of Stachybotrys atra.

Stachybotrys atra is the etiologic agent of stachybotryotoxicosis, and this fungus and its trichothecene mycotoxins were recently implicated in an outbreak of unexplained illness in homes. S. atra was grown on sterile rice, autoclaved, dried, and then aerosolized by acoustic vibration. The distribution of particles (mass and number) was monitored on an aerodynamic particle sizer interfaced with a computer. Dust was collected on preweighed glass-fiber filters and extracted with 90% aqueous methanol. Extracts were tested for the ability to inhibit protein synthesis in rat alveolar macrophages, the ability to inhibit the proliferation of mouse thymocytes, and the presence of specific trichothecene mycotoxins. Virtually all of the particles were less than 15 micron in aerodynamic diameter, and the mass median diameter was 5 micron. Thus, most of the particles were respirable. Microscopic analysis of the generated dust revealed that ca. 85% of the dust particles were conidia of S. atra, another 6% were hyphal fragments, and the remainder of the particles were unidentifiable. Thus, greater than 90% of the particles were of fungal origin. The extracts strongly inhibited protein synthesis and thymocyte proliferation. Purified satratoxin H was also highly toxic in the same systems. Each of the individual filters contained satratoxin H (average, 9.5 ng/mg of dust). Satratoxin G and trichoverrols A and B were found in lesser amounts in some, but not all, of the filters. The limit of analysis is ca. 50 ng. These results establish that the conidia of S. atra contain trichothecene mycotoxins. In view of the potent toxicity of the trichothecenes, the inhalation of aerosols containing high concentrations of these conidia could be a potential hazard to health.     

Particle size of airborne mouse crude and defined allergens

Laboratory animal allergy is a serious occupational diseases of many workers and scientists engaged in animal experimentation. Control measures depend upon characterization of allergens including airborne particles. This study measured the particle size of crude mouse urine and pelt aeroallergens generated in mouse housing rooms and compared them with mouse serum albumin, a defined major allergen. Allergens were detected by specific immunological methods. Most crude and defined allergens (74.5-86.4%) concentrated on a filter with a retention size greater than 7 microns. In distrubed air, allergen concentration increased 1.4 (albumin) to 5 (crude) fold and the proportion of small particles increased from 1.4% in calm air to 4.5% in distrubed air. This information on the generation and size distribution of aeroallergens will be important in the development of effective counter measures.     

Circadian rhythm of air-borne bacteria and dust particles in a mouse breeding room

The circadian rhythm of air-borne bacteria and dust particles in a mice breeding room was studied at 1-hour intervals on the first, third and fifth day after accommodation of the animals. The numbers of air-borne bacteria in the room increased day by day after accommodation, and showed a circadian rhythm which went down at about noon and rose with three peaks at about 20:00, 1:00 and 8:00. The numbers of dust particles tended to decrease from day to day, and they showed almost the same circadian rhythm as the air-borne bacteria. Correlations between air-borne bacteria and dust particles were not significant for each particle level on the first day, but were significant for all the particle levels of 0.3, 1, 2, and 5 microns at the third day, and were also significant at particles levels of 0.5 micron or more on the fifth day.     

An investigation of the presence of ultramicrocells in natural mineral water

The presence of 'ultramicrocells' in natural mineral water, capable of passing through a 0.2 micron filter, has been demonstrated. Filters allowing the greatest proportion of viable (culturable) cells to pass ranked in the order, 0.4 micron polycarbonate (5.02%) > 0.2 micron polycarbonate (0.02%) > or = 0.45 micron cellulose nitrate (0.02%) > 0.2 micron cellulose acetate (< 0.002%). Following incubation for 4 d at 22 degrees C, viable counts in filtered mineral water increased from < 2-8.7 x 10(2) cfu ml-1(-2).8 x 10(4)-1.9 x 10(6) cfu ml-1. Successive filtration/incubation cycles of mineral water increased the proportion of cells passing through a 0.2 micron cellulose acetate filter from < 0.003% to 0.11% and 0.69%, suggesting selection for 'ultramicrocells'. Cells isolated from this process and grown on liquid R2A medium were thin, Gram-negative rods, of 0.15-0.40 micron wide and 0.50-6.20 microns long. Membrane filtration techniques used for pathogen detection in mineral waters will not retain all the cells present. If pathogens are able to form ultramicrocells, these may go undetected.     

Filter efficiency of commercial face masks in capturing particles and airborne bacteria

The filter efficiency of seven kinds of commercial face mask for particles and airborne bacteria was tested in the wash room of a laboratory animal facility. The filter efficiency of the masks was 19 to 50%, as measured by the weight of particles with diameters below 10 micron, 22 to 71% for particles of the 0.3 micron level, 47 to 90% for the 1 micron level, and 90 to 99.6% for the 5 micron level. The filter efficiency for airborne bacteria was 35 to 81%. Among these even masks tested, glasswool surgery masks, three-sheet synthetic fiber masks with and without charcoal, and 28-sheet gauze masks with glass filter showed generally high efficiency, and single-sheet synthetic fiber masks, 18-sheet of gauze masks and gas masks showed low efficiency.     

Action of respiratory inhibitors on the electron transport system of Escherichia coli]

Bacteria of two strains of Escherichia coli (Q13 and MRE 600) were disintegrated by aluminium oxide. The influence of the respiratory inhibitors RF (a protein from reticulocytes), carboxin, Dexon (fungicides), thenoylftrifluoroacetone (TTFA), rotenone, antimycin A, myristic acid and monolaurin was tested on the succinate oxidase and the NADH oxidase system, respectively, of the membrane preparation obtained in this way as well as on the NADH oxidase activity of the cytosol. Among the inhibitors listed, only TTFA (5mM) inhibited the succinate oxidase system and Dexon (10 miconr), monolaurin (100 micron) and myristic acid (100 micron) inhibited the NADH oxidase system of the membranes. KCN (10 micron) inhibited both NADH oxidase systems. The inhibitory effects by monolaurin and myristic acid were prevent by human serum albumin and were markedly weaker than those on beef heart mitochondrial particles under similar conditions. The results argue for a divergent structure of the iron-sulphur proteins in the dehydrogenase regions of the electron transport system in comparison with animal and plant mitochondria and, moreover, confirm the specificity of RF and carboxin as well as the nature of Dexon as a group reagent on pyridine nucleotide dependent flavin enzymes.     

大型实验动物屏障设施环境微生物和尘埃粒子的动态监控

目的本实验以大型实验动物屏障设施为研究对象,分析屏障设施的环境微生物及尘埃粒子的动态变化规律及其相关影响因素。方法测定屏障设施各功能区域不同时间及不同工作状态下空气落下菌和直径≥0.3μm尘埃粒子的数量变化。结果屏障系统落下菌与尘埃粒子变化规律如下：屏障系统内空气落下菌与尘埃粒子在饲养工作后显著升高,喷雾消毒后明显降低;大、小鼠饲育室空气落下菌与尘埃数在凌晨时明显升高,而兔饲育室在凌晨时间段则较低;清洁走廊和污染走廊在工作状态时细菌含量明显上升,非工作状态时细菌含量一直处于较低的水平。结论实验动物屏障系统的环境微生物与尘埃粒子的动态数量变化与动物品种、空气消毒、人员进出及动物室内的饲养操作等有关。     

Further characterization of the effects of alpha-1-acid glycoprotein on the passage of human erythrocytes through micropores

Effects of human alpha-1-acid glycoprotein (AG) on the passage of human red blood cell(s) (RBC) through membrane filters with micropores were examined in vitro. RBCs, with a mean major diameter of 7.2 micron, that had been suspended at 1% in physiological phosphate-buffered saline (PBS), were filtered through membrane filters of various pore diameters under positive pressure. The percentages of cells that passed through the micropores and of cells hemolyzed during filtration were determined. RBCs suspended in PBS did not pass through micropores that had an average pore diameter of 3 micron; instead hemolysis took place. Neither temperature nor applied pressure affected cell passage; but when AG at 0.1 mg/ml or above was added to an RBC-suspension, it promoted cell passage through the 3 micron micropores and reduced the degree of hemolysis. The effects of AG were dose dependent up to a concentration of 0.5 mg/ml. The addition of AG to an RBC-suspension that contained 90% human serum had the same additive effects. Washing AG-treated RBCs with normal saline produced a marked decrease in cell passage through the 3 micron pores. Fluorescence antibody staining revealed that the exogenous AG was localized on the membrane surface of the RBCs. Our results suggest that the AG bound to the surface of the RBCs acts as a lubricant between the RBCs and the wall of the micropore; this would facilitate RBC-passage through the micropores.     

Aerosols containing Legionella pneumophila generated by shower heads and hot-water faucets

Shower heads and hot-water faucets containing Legionella pneumophila were evaluated for aerosolization of the organism with a multistage cascade impaction air sampler. Air was collected above two shower doors and from the same rooms approximately 3 ft (91 cm) from the shower doors while the hot water was running. Low numbers (3 to 5 CFU/15 ft3 [0.43 m3] of air) of L. pneumophila were recovered above both shower doors, but none was recovered from the air in either room outside the shower door. Approximately 90% (7 of 8 CFU) of the L. pneumophila recovered were trapped in aerosol particles between 1 and 5 micron in diameter. Air was collected 1 to 3 ft (30 to 91 cm) from 14 sinks while the hot water was running. Low numbers (1 to 5 CFU/15 ft3 of air) were recovered from 6 of 19 air samples obtained. Approximately 50% (6 of 13 CFU) of the organisms recovered were trapped in aerosol particles between 1 and 8 microns in diameter. Shower heads and hot-water taps containing L. pneumophila can aerosolize low numbers of the organism during routine use. The aerosol particle size is small enough to penetrate to the lower human respiratory system. Thus, these sites may be implicated as a means of transmission of L. pneumophila from potable water to the patient.     

Aerosols containing Legionella pneumophila generated by shower heads and hot-water faucets.

Shower heads and hot-water faucets containing Legionella pneumophila were evaluated for aerosolization of the organism with a multistage cascade impaction air sampler. Air was collected above two shower doors and from the same rooms approximately 3 ft (91 cm) from the shower doors while the hot water was running. Low numbers (3 to 5 CFU/15 ft3 [0.43 m3] of air) of L. pneumophila were recovered above both shower doors, but none was recovered from the air in either room outside the shower door. Approximately 90% (7 of 8 CFU) of the L. pneumophila recovered were trapped in aerosol particles between 1 and 5 micron in diameter. Air was collected 1 to 3 ft (30 to 91 cm) from 14 sinks while the hot water was running. Low numbers (1 to 5 CFU/15 ft3 of air) were recovered from 6 of 19 air samples obtained. Approximately 50% (6 of 13 CFU) of the organisms recovered were trapped in aerosol particles between 1 and 8 microns in diameter. Shower heads and hot-water taps containing L. pneumophila can aerosolize low numbers of the organism during routine use. The aerosol particle size is small enough to penetrate to the lower human respiratory system. Thus, these sites may be implicated as a means of transmission of L. pneumophila from potable water to the patient.     

Response of colony-forming units-spleen to heavy charged particles

Survival of colony-forming units-spleen (CFU-S) was measured after single doses of photons or heavy charged particles from the BEVALAC. The purposes were to define the radiosensitivity to heavy ions used medically and to evaluate relationships between relative biological effectiveness (RBE) and dose-averaged linear energy transfer (LET infinity). In in vitro irradiation experiments. CFU-S suspensions were exposed to 220 kVp X rays or to 20Ne (372 MeV/micron) or 40Ar (447 MeV/micron) particles in the plateau portion of the Bragg curve. In in vivo irradiation experiments, donor mice from which CFU-S were harvested were exposed to 12C (400 MeV/micron). 20Ne (400 or 670 MeV/micron), or 40Ar (570 MeV/micron) particles in Bragg peaks spread to 4 or 10 cm by spiral ridge filters. Based on RBE at 10 survival, the maximum RBE of 2.1 was observed for 40Ar particles characterized by an LET infinity of approximately 100 keV/micron. Lower RBEs were determined at lower or higher estimated values of LET infinity and ranged from 1.1 for low energy 40Ar particles to 1.5-1.6 for low energy 12C and 20Ne. The responses of CFU-S are compared with responses of other model systems to heavy charged particles and with the reported sensitivity of CFU-S to neutrons of various energies. The maximum RBE reported here, 2.1 for high energy 40Ar particles, is somewhat lower than values reported for fission-spectrum neutrons, and is appreciably lower than values for monoenergetic 0.43-1.8 MeV neutrons. Low energy 12C and 20Ne particles have RBEs in the range of values reported for 14.7 MeV neutrons.     

Monoclonal antibodies to the major feline allergen Fel d I. II. Single step affinity purification of Fel d I, N-terminal sequence analysis, and development of a sensitive two-site immunoassay to assess Fel d I exposure

Two mAb were used to develop new techniques for the purification and quantitation of the major feline salivary allergen, Felis domesticus allergen I (Fel d I). The allergen was purified from aqueous house dust extract with a high Fel d I content by affinity chromatography over a monoclonal immunosorbent and elution with 4 mM HCl, pH 2.5. This single step procedure gave 40 to 50% recovery of 90% pure allergen which, following final purification by size exclusion HPLC, showed a single line on immunodiffusion and crossed immunoelectrophoresis against monospecific anti-Fel d I and polyclonal anti-cat dander antibodies. The m.w. of native Fel d I was 39,000 on size exclusion HPLC, and 17,000 under nonreducing conditions on gel electrophoresis. The N-terminal amino acid sequence (33 residues) showed no homology with other known protein sequences. The combination of the SDS-PAGE and N-terminal sequence data suggests that Fel d I is a non-covalently linked homodimer. A two-site RIA was developed using mAb directed against different epitopes on Fel d I. This assay was species-specific, highly sensitive (0.0004 U/ml), and showed an excellent correlation with a polyclonal inhibition RIA (n = 27, r = 0.93, p less than 0.001). Cat allergen extracts used for immediate skin tests showed marked differences in Fel d I content (from 0.1 to 30 U/ml). Consistently high Fel d I levels were found at monthly intervals in six dust samples from four houses with cats (10 to 100 U/g of dust). Comparisons of Fel d I and mite and pollen allergen levels showed that house dust can contain greater than 100 micrograms/g of either of these allergens and is a potent source of foreign environmental antigens. Monoclonal affinity chromatography provides a major breakthrough in the purification of Fel d I, from a source material that would otherwise have been considered impossible (house dust). The mAb assay for Fel d I is both more sensitive and more easily standardized than existing techniques. These techniques will allow full structural and antigenic analysis of Fel d I and more detailed studies on the relationship between cat antigen exposure and the development of asthma.     

Reducing exposure to laboratory animal allergens

Laboratory animal allergy is a serious health problem. We examined several possible allergen-reducing strategies that might be effective in the working mouse room. Ambient allergen concentrations were measured when mice were maintained under several conditions: conventional housing versus ventilated cage racks operated under negative or positive pressure. We found that housing mice in ventilated cages operated under negative pressure and using ventilated changing tables reduced ambient mouse allergen (Mus m 1) concentrations tenfold, compared with values when mice were housed in conventional caging and using a conventional (non-ventilated) changing table. Housing mice in positively pressurized cages versus conventional cages did not reduce ambient allergen values. Cleaning mouse rooms at an accelerated frequency also did not reduce ambient Mus m 1 concentration. We also quantified ambient allergen values in several areas of The Jackson Laboratory. A facility-wide survey of Mus m 1 concentrations indicated that allergen concentrations were undetectable in control areas, but ranged from a mean (+/- SEM) 0.11 +/- 0.02 ng/m3 to 5.40 +/- 0.30 ng/m3 in mouse rooms with different cage types. The percentage of animal caretakers reporting allergy symptoms correlated significantly with ambient allergen concentrations: 12.9% reported symptoms in the rooms with the lowest allergen concentration (0.14 +/- 0.02 ng/m3), but 45.9% reported symptoms in rooms with the highest concentration (2.3 +/- 0.4 ng/m3). These data indicate that existing technology can significantly reduce exposure to laboratory animal allergens and improve the health of animal caretakers.     

Immunological evaluation of urinary trypsin inhibitors in blood and urine: Role of N- & O-linked glycoproteins

Urinary trypsin inhibitors (uTi) suppress serine proteases during inflammation. After liberation from proinhibitors (P-alpha-I and I-alpha-I) by the white blood cell (WBC) response, uTi readily pass through the kidneys into urine. A key uTi, bikunin, is attached to O-linked and N-linked glycoconjugates. Recently, uTi inhibitors, called uristatins, were found to lack the O-linked glycoconjugates. Monoclonal antibodies were produced using purified uristatin and screened for binding differences to uristatin, bikunin, P-α-I, and I-α-I. Antibody-binding patterns were characterized using immunoaffinity binding onto protein-chip surfaces and analysis by Surface Enhanced Laser Desorption/Ionization mass spectrometry (SELDI), using specimens from patients and from purified uTi standards. Antibodies were developed and used in an enzyme-linked immunosorbent assay (ELISA) method for uTi measurement in urine and plasma specimens. ELISA was performed on specimens from normal, presumed healthy, controls and from patients who had been screened for inflammation using a high sensitivity C-reactive protein (CRP) test and a complete blood count (CBC). Polyclonal antibody against uTi showed cross-reactivity with the Tamm–Horsfall protein (THP) and with proinhibitors. Screening of anti-uTi monoclonal antibodies (Mab) revealed antibodies that did not cross-react with either of the above, thus providing a tool to measure both uristatin and bikunin in urine with Mab 3G5 and in plasma with Mab 5D11. The monoclonal antibody 5D11 cross-reacts with specific N-linked glycoconjugates of uristatin present in plasma. In ca 96% of healthy adults, uTi were present at <12 mg/l in urine and <4 mg/l in plasma. We also found that patients with an inflammation and a CRP of >2.0 mg/l had higher urinary concentrations of uTi than the control population in every subject. Free uristatin and bikunin pass readily into urine and are primarily bound to heavy chains that constitute the proinhibitor form in plasma.     

实验动物致敏研究进展

实验动物致敏（laboratory Animal Allergy,LAA）,是一种职业过敏性疾病,造成人的呼吸道及皮肤发生炎症。该病在国外研究较多,过敏源是动物皮毛、尿液、唾液中的一类酸性小分子蛋白质;可以通过皮肤试验、放射过敏吸附试验及ELISA等方法检测易感人员。对易感人员可以通过控制环境中的过敏原来保护。目前国内尚无专门机构研究此病症,笔者综述了该职业性疾病的症状、机理、控制方法及国内外对该病症认识上的差异。     

A quantitative study of intramembrane changes during cell junctional breakdown in the dystrophic rat retinal pigment epithelium

Previous electron microscope freeze-fracture and tracer studies have revealed that intercellular junctions in the retinal pigment epithelium (RPE) of Royal College of Surgeons (RCS) rats with inherited retinal dystrophy [5] break down between three and six postnatal weeks [6, 7]. In this study quantitative computer techniques were used to analyze the freeze-fracture changes in the dystrophic RPE. The following parameters were measured: length of tight junctional strands/micron2; number of tight junctional strand anastomoses/micron2; number of gap junctional aggregates/micron2; area of gap junctional aggregates/micron2; and density of background intramembrane particles/micron2. At three postnatal weeks, the dystrophic junctional complex membrane is similar to normal, but at 10 weeks and later there are dramatic decreases in tight junctional strand length/micron2 and number of anastomoses/micron2, as well as in the number/micron2 and area of gap junctions/micron2, while the density of background particles/micron2 is dramatically increased. Correlational analysis revealed that changes in gap and tight junctions were significantly related to each other and to the increase in background particle density. The diameter of background particles within the normal and post-breakdown dystrophic junctions was measured in order to see whether the dispersal of gap and tight junctional particles (8-10 nm) into the surrounding membrane contributes to the increased particle density. These measures showed that background particles in all size ranges were more numerous in the dystrophic RPE, but that the largest increase was in the smallest diameter particles (6-7 nm). Thus, while gap and tight junctional sized particles contribute to the increase, particles from other sources may also be involved. Particle density of apical and basal membranes in the normal and in the 10 week and older dystrophic RPE was analyzed to study the effects of tight junctional breakdown on the distribution of intramembrane particles. These measures showed that particle density was greater basally than apically in the normal RPE and that particle density in both membranes decreased slightly in the dystrophic RPE, but that their ratio remained unchanged. It has been shown previously that even a single intact tight junctional strand is sufficient to maintain differences in particle density between apical and basal surfaces [14, 15] and in the majority of abnormal dystrophic junctional complexes at least one tight junctional strand remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)