Evaluation of different methods to detect microbial hygiene indicators relevant in the dairy industry.

It is estimated that 19% of the total food loss from retail, food service, and households comes from dairy products. A portion of this loss may be attributed to premature spoilage of products due to lapses in sanitation and postpasteurization contamination at the processing level. Bacterial groups including coliforms, Enterobacteriaceae (EB), and total gram-negative organisms represent indicators of poor sanitation or postpasteurization contamination in dairy products worldwide. Although Petrifilms (3M, St. Paul, MN) and traditional selective media are commonly used for the testing of these indicator organism groups throughout the US dairy industry, new rapid methods are also being developed. This project was designed to evaluate the ability of different methods to detect coliforms, EB, and other gram-negative organisms isolated from various dairy products and dairy processing environments. Using the Food Microbe Tracker database, a collection of 211 coliform, EB, and gram-negative bacterial isolates representing 25 genera associated with dairy products was assembled for this study. We tested the selected isolates in pure culture (at levels of approximately 15 to 300 cells/test) to evaluate the ability of 3M Coliform Petrifilm to detect coliforms, 3M Enterobacteriaceae Petrifilm, violet red bile glucose agar, and an alternative flow cytometry-based method (bioMérieux D-Count, Marcy-l'Étoile, France) to detect EB, and crystal violet tetrazolium agar to detect total gram-negative organisms. Of the 211 gram-negative isolates tested, 82% (174/211) had characteristic growth on crystal violet tetrazolium agar. Within this set of 211 gram-negative organisms, 175 isolates representing 19 EB genera were screened for detection using EB selective/differential testing methods. We observed positive results for 96% (168/175), 90% (158/175), and 86% (151/175) of EB isolates when tested on EB Petrifilm, violet red bile glucose agar, and D-Count, respectively; optimization of the cut-off thresholds for the D-Count may further improve its sensitivity and specificity, but will require additional data and may vary in food matrices. Additionally, 74% (129/175) of the EB isolates tested positive as coliforms. The data obtained from this study identify differences in detection between 5 microbial hygiene indicator tests and highlight the benefits of EB and total gram-negative testing methods.

Paratiroidectomía en pacientes renales crónicos en Argentina: estudios prequirúrgicos, tipos de cirugía, recurrencia y persistencia / Parathyroidectomy in chronic kidney disease patients in Argentina: pre surgical studies, types of surgery, recurrence and persistence

La paratiroidectomía (PTx) es el tratamiento de elección en pacientes con HPT 2º severo, refractario al tratamiento médico. Se cuenta con muy poca información en Argentina de este procedimiento, por lo cual se realizó este estudio. Material y Métodos: Se incluyeron 255 pacientes con PTx entre el año 2003 al 2007 de un registro voluntario. Se evaluaron los estudios de localización prequirúrgicos, de laboratorio de metabolismo fosfocálcico previo y posterior a la cirugía y el tipo de técnica quirúrgica utilizada. Se analizó la persistencia y recidiva del HPT postcirugía. Resultados: La tasa de PTx fue de 2,7/1000 pacientes año. 83% de los pacientes tuvieron ecografía de cuello y 59% Sesta Mibi con Tc 99. Hubo una correlación positiva (p<0.001) entre el número de glándulas detectadas por ecografía y Sesta Mibi. La paratiroidectomía realizada fue: subtotal en 77%, total con autoimplante en 14% y total sin autoimplante en 9%. Hubo descensos significativos de Ca y P, fosfatasa alcalina y PTH (1744 ± 788 pg/ml a 247 ±450 pg/ml; p<0.0001) postcirugía. A los 2,4 ±2,5 meses de la PTx, el 72% de los pacientes tenía PTH <250 pg/ml, 19,8% tenía persistencia y 8,3% había recidivado. De acuerdo al tipo de cirugía la persistencia y recidiva fueron para PTx subtotal 22% y 8,3%, PTx total con implante 11% y 11% y PTx total sin autoimplante 13% y 4% respectivamente. La realización de Sesta Mibi no influyó en los resultados de la PTx. No se observaron diferencias entre los centros en relación con persistencia y recidiva. Conclusiones: La tasa de PTx fue muy baja, la ecografía fue el método de localización prequirúrgico preferido y la PTX subtotal la técnica quirúrgica más utilizada. La PTx fue exitosa en la mayoría de los pacientes y la persistencia y recidiva no estuvieron relacionadas con la técnica.

Parathyroidectomy (PTx) is the selecte treatment for patients with severe secondary hyperparathyroidism, refractory to medical treatment. There is not enough information about this procedure in Argentina, that is the reason why we performed this study. Material and Methods: 255 patients with PTx were included from the year 2003 to 2007 on a voluntary register. Studies of pre-surgical localization, phosphocalcic metabolism laboratories before and after surgery were evaluated, and the type of surgical technique used. The persistence and recurrence of post-surgical hyperparathyroidism was analyzed. Results: The PTx rate was 2,7/1000 patients year. 83% of the patients had neck echography and 59% Sestamibi scans with Tc 99. There was a positive correlation (p<0,001) between the number of detected glands by echography and Sestamibi. The parathyroidectomy performed was: subtotal in 77%, total with self-implant in 14% and total without self-implant in 9%. There were significant falls of Ca and P, Alkaline Phosphatase and PTH (1744±788 pg/ml to 247±450 pg/ml; p<0.0001) post-surgical. 2.4 ±2,5 months after the PTx, 72% of patients had PTH <250 pg/ml, 19,8% had persistence and 8,3% had recurrence. According to the type of surgery, the persistence and recurrence were for subtotal PTx 22% and 8,3%, total PTx with implant 11% and 11%, and total PTx without selfimplant 13% and 4% respectively. The performance of the Sestamibi scan did not affect the PTx results. No noticeable differences were observed among the centers for persistence and recurrence. Conclusions: The PTx rate was very low, echography was the preferred method of pre-surgical localization, and subtotal PTx was the most used surgical technique. PTx was successful in most of the patients, and persistence and recurrence were not related to the technique.

Polymeric nanocapsules ultra stable in complex biological media.

Non-specific protein adsorption from complex biological media, especially from blood plasma, is an urgent challenge for the application of nanoparticles as delivery systems, diagnostics, and other biomedical application. Nanocapsules (NC) prepared from FDA-approved degradable poly(ɛ-caprolactone) shell and Mygliol 812(®) oil in the core were coated with mono-methoxy terminated oligo(ethylene glycol) methacrylate (poly(MeOEGMA)) polymer brush layers with a well-controlled thickness at the nanometer scale up to 350 nm using surface initiated atom transfer radical polymerization in water or phosphate buffered saline. Incubation of uncoated NC with human serum albumin solution, fetal bovine serum, or human blood plasma resulted in fast aggregation observed by dynamic light scattering as an increase in diameter of particles present in the solutions. Conversely, these biological fluids affected only marginally the size distribution of the NC coated with a 60 nm thick poly(MeOEGMA) layer. The high suspension stability of the coated NC in complex biological fluids was related to the suppressed deposition of proteins from these fluids observed by surface plasmon resonance (SPR) on analogous poly(MeOEGMA) layer prepared on flat surfaces of SPR chips.

Interaction of blood plasma with antifouling surfaces.

Nonspecific adsorption of proteins is a crucial problem in the detection of analytes in complex biological media by affinity sensors operating with label-free detection. We modified the gold surface of surface plasmon resonance (SPR) sensors with three types of promising antifouling coatings: self-assembled monolayers (SAM)s of alkanethiolates terminated with diethylene glycol and carboxylic groups, poly(ethylene glycol) (PEG) grafted onto the SAMs, and zwitterionic polymer brushes of poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), and poly(phosphorylcholine methacrylate). Using SPR, we compared the efficacy of the coatings to reduce nonspecific adsorption from human blood plasma and from single-protein solutions of human serum albumin, immunoglobulin G, fibrinogen, and lysozyme. There was no direct relationship between values of water contact angles and plasma deposition on the coated surfaces. A rather high plasma deposition on SAMs was decreased by grafting PEG chains. Fouling on PEG was observed only from plasma fractions containing proteins with molecular mass higher than 350 000 Da. The adsorption kinetics from plasma collected from different healthy donors differed. Poly(carboxybetaine methacrylate) completely prevented the deposition from plasma, but the other more hydrophilic zwitterionic polymers prevented single-protein adsorption but did not prevent plasma deposition. The results suggest that neither wettability nor adsorption of the main plasma proteins was the main indicator of deposition from blood plasma.

[Importance of the "adequate blood phosphorus" concept as a risk factor for hyperphosphatemia]. / Importancia del concepto "fosfatemia adecuada" como factor de riesgo de hiperfosfatemia.

Hyperphosphatemia is an important risk factor of secondary hyperparathyroidism and extraosseous calcifications in chronic renal failure patients. In this study our hypothesis is that physicians misconception of adequate phosphatemia is a risk factor for hyperphosphatemia. In 1999 GEMOR sent a renal osteodystrophy inquiry to different hemodialysis centers in Argentina. It included 80 dialysis centers in 17 Argentinian provinces. The enquire had 33 questions about renal osteodystrophy. Here we report the section related to phosphorous metabolism. We obtained responses from 80 dialysis centers (4,512 dialysis patients), which represents about 24% of Argentinian dialysis centers. Physicians considered phosphorous levels between 4.5 to 5.5 mg/dl in 83.5% of centers as adequate, and between 5.5 to 6.5 mg/dl in 10.1%. Five out of 77 centers reported that they had no patients with hyperphosphatemia. The percentage of hemodialysis patients that had more than 6 mg/dl in each center was 28.8 +/- 15.9%. Those centers that aimed for phosphatemia between 5.5 and 6.5 mg/dl, had a higher percentage of patients with phosphatemia above 6 mg/dl than those aiming for between 4.5 and 5.5 mg/dl (42.8 +/- 16.7 vs 27.1 +/- 15.2% respectively, p = 0.007), and had higher mean of phosphatemia (6.4 +/- 0.7 vs 5.3 +/- 0.7 mg/dl respectively, p = 0.0001), than the last group. In conclusion, a higher mean phosphate level was obtained in hemodialysis centers where physicians considered higher pre-dialysis target levels. Some centers had no patients with hyperphosphatemia (neglect or good control?).

Importancia del concepto «fosfatemia adecuada» como factor de riesgo de hiperfosfatemia / Physician misconception of “adequate phosphatemia” may be a determinant factor for hyperphosphatemia

En 1999 GEMOR realizó una encuesta entre centros de diálisis de Argentina para conocer la realidad de dicha patología en el país. Presentamos los resultados relacionados al fósforo. Participaron 80 centros de diálisis (24% de los centros nacionales), donde dializaban 4.512 pacientes (34% del país). El 95% determinaban Calcio y Fósforo sérico en forma mensual. El 83,5% pretendía en sus pacientes una fosfatemia entre 4,5 y 5,5 mg/dl, mientras que en el 10,1% el objetivo estaba entre 5,5 y 6,5 mg/dl. La media porcentual de fosfatemia superior a 6 mg/dl fue del 28,8 ± 15,9%. Cinco de 77 centros reportaron que no tenían pacientes hiperfosfatémicos en la última determinación mensual. Los que pretendían una fosfatemia entre 5,5 y 6,5 mg/dl, tenían un porcentaje de pacientes con fosfatemia mayor de 6 mg/dl superior a aquellos que pretendían entre 4,5 y 5,5 mg/dl (42,8 ± 16,7 vs 27,1 ± 15,2% respectivamente, p = 0,007). Mientras que la media de fosfatemia también fue superior (6,4 ± 0,7 vs 5,3 ± 0,7 mg/dl respectivamente, p = 0,0001). Esto sugiere que los centros donde se pretendía mayor fosfatemia tenían más casos de hiperfosfatemia. Existen centros sin pacientes hiperfosfatémicos. Esto supone o un buen control de la fosfatemia o resultados de laboratorio «falsos negativos» (AU)

Hyperphosphatemia is an important risk factor of secondary hyperparathyroidism and extraosseous calcifications in chronic renal failure patients. In this study our hypothesis is that physicians misconception of adequate phosphatemia is a risk factor for hyperphosphatemia. In 1999 GEMOR sent a renal osteodystrophy inquiry to different hemodialysis centers in Argentina. It included 80 dialysis centers in 17 Argentinian provinces. The enquire had 33 questions about renal osteodystrophy. Here we report the section related to phosphorous metabolism. We obtained responses from 80 dialysis centers (4,512 dialysis patients), which represents about 24% of Argentinian dialysis centers. Physicians considered phosphorous levels between 4.5 to 5.5 mg/dl in 83.5% of centers as adequate, and between 5.5 to 6.5 mg/dl in 10.1%. Five out of 77 centers reported that they had no patients with hyperphosphatemia. The percentage of hemodialysis patients that had more than 6 mg/dl in each center was 28.8 ± 15.9%. Those centers that aimed for phosphatemia between 5.5 and 6.5 mg/dl, had a higher percentage of patients with phosphatemia above 6 mg/dl than those aiming for between 4.5 and 5.5 mg/dl (42.8 ± 16.7 vs 27.1 ± 15.2% respectively, p = 0.007), and had higher mean of phosphatemia (6.4 ± 0.7 vs 5.3 ± 0.7 mg/dl respectively, p = 0.0001), than the last group. In conclusion, a higher mean phosphate level was obtained in hemodialysis centers where physicians considered higher pre-dialysis target levels. Some centers had no patients with hyperphosphatemia (neglect or good control?) (AU)

Frequency of factor V(Leiden) and prothrombin G20210A in placentas and their relationship with placental lesions.

The most common hereditary hypercoaguable states are factor V(Leiden) (FVL) and prothrombin mutations (PRO). FVL and PRO present with an incidence of approximately 5% in a heterogeneous population, and 45% to 63% of the thrombophilic population. The frequency of these mutations in the fetal population and their clinical importance is unknown. Fetal side thromboembolic events (FST) include congenital stroke and renal vein thromboses. In some cases, FST can be diagnosed by placental histopathology when avascular (infarcted) villi are present in a patent maternal vascular space. FST can present as placenta-fetal-vascular or fetal-visceral-vascular lesions. Causes include vascular damage from cord compression or inflammation, but most remain unclear. Potential causes of FST include FVL and PRO. We describe the incidence of FVL and PRO from a prospective group of 169 consecutive placentas and in a retrospective group of archived placentas diagnosed with placental FST. One each of FVL and PRO heterozygosity was found in the prospective set (< 1% incidence for each). Five prospective placentas were diagnosed with placental FST, for an incidence of 3%; all were wild-type for FLV and PRO. Twenty-seven of 65 archived FST cases had analyzable DNA to find 5 FVL heterozygotes (18.5%); all were wild-type for PRO. Twenty-one of 65 retrospective archived controls analyzable found 1 case of FVL heterozygosity (< 5%). We find that the frequency of FVL and PRO may be decreased in the pregnant population but increased in cases of placental FST. Because factor V Leiden heterozygosity carries an increased risk for thrombotic complications, we suggest placental diagnosis of fetal side thromboemboli warrants clinical evaluation for FVL in infant and potentially the parents.

Aluminum exposure in chronic renal failure in iberoamerica at the end of the 1990s: overview and perspectives.

Epidemic aluminum neurotoxicity has virtually disappeared in the dialysis population; however, sporadic toxic effects caused by contamination of water with aluminum are still reported. In this review, the current situation in Iberoamerica is analyzed. Exposure to aluminum through dialysate shows considerable geographical differences even within the same country, including seasonal variability. Sometimes the tap water showed very high aluminum content that does not permit the water treatment system to efficiently remove all the aluminum, forcing the use of water treatment systems with a double reverse-osmosis filter on line. The use of adequate water treatment systems and a correct control policy has improved the quality of the dialysate, minimizing the aluminum exposure. However, an additional problem in Iberoamerica is the difficulty to obtain aluminum-free concentrates for the preparation of the final dialysis solution. Aluminum still seems to be implicated in a great percentage of symptomatic low-bone remodeling lesions in South America compared with Europe, demonstrating that exposure to aluminum through dialysate is still a cause of concern in some areas of the world.

Prospective comparison of direct immunofluorescence and conventional staining methods for detection of Giardia and Cryptosporidium spp. in human fecal specimens.

In a prospective comparative study, 2,696 consecutive fresh stool specimens over the course of 1 year were examined for Giardia lamblia and Cryptosporidium parvum by using a direct immunofluorescent-monoclonal antibody stain (for unspun specimens) and conventional staining methods (chlorazol black E for Giardia cysts and modified Kinyoun acid-fast for Cryptosporidium oocysts). The direct immunofluorescent-monoclonal antibody method resulted in a significantly increased detection rate for both giardia (118 versus 79 specimens, 49.4%; P = 0.006) and cryptosporidia (39 versus 23 specimens, 69.6%; P = 0.055).

Embryonic cell death patterns associated with nitrofen-induced congenital diaphragmatic hernia.

The offspring of pregnant Sprague-Dawley rats exposed to nitrofen on gestational day 9.5 develop left-sided congenital diaphragmatic hernia (CDH). Twenty-four hours after treatment, on day 10.5, supravital staining with Nile blue sulfate and histological examination showed bilateral excessive cell death in cervical somites 2 through 4. After 48 hours, on day 11.5, cell death was absent in the cervical somites but was apparent in the mesoderm adjacent to the somites in the septum transversum and in the developing sympathetic ganglia adjacent to the dorsal aortae. Cell death was not apparent in the foregut or lung primordia on either day 10.5 or 11.5. The incidence of nitrofen-exposed embryos with such patterns of cell death closely paralleled that of left-sided CDH in similarly treated day 21.5 fetuses. Control animals treated with olive oil had normal programmed cell death patterns in the regions of interest and had no evidence of CDH on day 21.5. It is possible that these patterns of excessive cell death early in gestation may play a role in the genesis of diaphragmatic hernia. Mesoderm derived from cervical somites 3 through 5 contributes to the diaphragmatic anlage and forms the major portion of the muscle of the diaphragm. Because nitrofen damages mesodermal cell populations in cervical somites 2 through 4 and in the mesenchyme adjacent to the septum transversum 24 to 48 hours after administration, the authors propose that damage to these populations may reduce progenitor cells needed to populate the diaphragmatic anlage, thereby hindering pleuro-peritoneal canal closure.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell death and cell cycle perturbation in the developmental toxicity of the demethylating agent, 5-aza-2'-deoxycytidine.

DNA methylation is a probable mechanism for regulating gene expression, and alterations in methylation may significantly affect embryonic development. We administered the cytidine analogue 5-aza-2'-deoxycytidine (dAZA), a specific and potent demethylator of DNA, to pregnant mice to determine its teratogenicity and effects on embryonic cell death and cell cycle. Groups of females were dosed intraperitoneally on gestation day 10 with doses of 0.05-3 mg/kg dAZA and killed at 4, 8, or 28 hr later. Two embryos per litter were immediately stained with Nile blue sulfate (NBS) to identify areas of cell death; the remaining embryos were frozen and stored for subsequent flow cytometric (FCM) analysis of the cellular DNA synthetic cycle in limb buds. A dose-related accumulation of cells in the S and G2/M phases was observed at 4 and 8 hr after maternal dosing. S-phase accumulation was the most sensitive indicator of effect; a dose-related increase in the percentage of hindlimb bud cells in S-phase was evident at all dosages 4 hr after maternal dosing. By 28 hr postdosing, a normal cell cycle phase distribution was observed at doses of < 0.3 mg/kg. However, cell cycle perturbations persisted at higher dosages. NBS staining demonstrated increased cell death in areas of rapid cell division, indicative of replication-associated cytotoxicity, at doses of > or = 0.1 mg/kg. Observation of litters from additional dams killed at term revealed that at dosages of > or = 0.3 mg/kg, cleft palate and hindlimb defects were significantly elevated. In addition, above 0.3 mg/kg, fetal weight was significantly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of CA 72-4 (TAG-72) in the fluid contents of pancreatic cysts. A new marker to distinguish malignant pancreatic cystic tumors from benign neoplasms and pseudocysts.

OBJECTIVE: The authors evaluated cyst fluid CA 72-4 as a tumor marker in the differential diagnosis of pancreatic cystic lesions. SUMMARY BACKGROUND DATA: Pancreatic cystic lesions include inflammatory pseudocysts, serious cystadenomas, and mucinous tumors. Mucinous tumors can be further subdivided into mucinous cystadenocarcinomas and premalignant mucinous cystic neoplasms. The clinical and radiologic features of these lesions are unreliable to make a preoperative diagnosis of these diagnostically difficult lesions. Analysis of aspirated cyst fluid was proposed as an aid to making the preoperative differential diagnosis. Currently, a number of parameters have been reported as useful markers in cyst fluid aspirates, including the tumor markers carcinoembryonic antigen and CA 15.3, enzymes (amylase, lipase, and amylase isoenzymes), relative viscosity, and cytologic analysis. However, owing to the rarity of pancreatic cystic tumors, experience with cyst fluid analysis is limited. To define additional markers that might be useful in the differential diagnosis of pancreatic cysts, the authors measured the tumor-associated glycoprotein 72 (TAG-72) in aspirates from 19 pancreatic cystic lesions. METHODS: Cyst fluid from 19 pancreatic cysts was obtained by needle aspiration. The tumor marker TAG-72 was measured by a commercial (CA 72-4) immunoassay. RESULTS: Cyst fluid CA 72-4 levels in mucinous cystadenocarcinomas were markedly elevated (mean, 10,027 U/mL; range, 780 to 34,853 U/mL) compared with that in pseudocysts (mean, 3.8 U/mL; range, < 3 to 5.7 U/mL) and serous cystadenomas (mean and range, < 3 U/mL; p < 0.001). The level of CA 72-4 in benign mucinous cystic neoplasms was intermediate (mean, 44.2 U/mL; range, < 3 to 137 U/mL), but it was statistically different from either carcinomas (p = 0.009) or benign cysts (p < 0.001).

Pathogenesis of ethanol-induced limb reduction defects in mice.

Acute administration of dosages of 2.5, 2.8, or 2.9 g/kg of ethanol to pregnant C57BL/6J mice on gestational day 9 1/4 resulted in major malformations of the forelimb including postaxial ectrodactyly, preaxial syndactyly, and reduction defects involving intermediate digits. The incidence and severity of these defects was positively correlated with dosage. Sidedness of the defects was also dose-dependent. In affected embryos, excessive amounts of cell death were notable within 5-9 hr of treatment initiation in selected cell populations. Cell death was primarily distributed in two regions of the developing limb bud--a ventrodistal ectodermal cell population (apical ectodermal ridge) and a proximal mesenchymal cell population. The patterns of cell death observed appear to be pathogenically related to the limb defects noted at later stages. In particular, it would appear that the deficiencies in the apical ectodermal ridge resulting from ethanol-induced cell death can account for virtually all the subsequent limb defects.

Pathogenesis of retinoid-induced hindbrain malformations in an experimental model.

Among the findings associated with the human Retinoic Acid Embryopathy are hindbrain defects including the Arnold-Chiari malformation. The human Arnold-Chiari malformation (ACM) is a malformation complex where the cardinal feature is herniation of the caudal hindbrain into the vertebral column; it is frequently accompanied by lumbosacral myelorachischisis and hydrocephalus. Mice exposed to all-trans-retinoic acid or etretinate on day 8.25 of pregnancy, produce offspring with hindbrain herniation and caudal lumbosacral myelorachischisis in addition to a variety of other craniofacial and caudal malformations. Several experimental animals were observed to lack the caudal myelorachischisis proving that this lesion is not required to generate hindbrain herniation. We provide evidence that the cranial malformations, including hindbrain herniation, result from primary damage to the neural crest and the rhombencephalon. The vulnerability of these sites appears to be correlated with the presence of normal physiological cell death. While these experimental animals differ in many respects from the typical human Arnold-Chiari malformation, they may provide some insight into the pathogenesis of the latter.

Relationship of cell death to cyclophosphamide-induced ocular malformations.

C57BL/6J mice were used to study the ocular teratogenic effects of cyclophosphamide administered to pregnant females on d 9 of pregnancy at a dose of 5 mg/kg body weight. Nile blue staining demonstrated increased cell death at the base of the optic stalk, in the optic vesicle, and in the perivesicular mesenchyme in treated embryos. Malformations studied at gestational d 11 and 16 by light and scanning electron microscopy included microphthalmos, microphakia, and aphakia and were predictable based upon patterns of increased cell death. These anomalies are similar to those reported with exposure to ethanol or isotretinoin on gestational d 7.

Cyclophosphamide teratogenesis: evidence for compensatory responses to induced cellular toxicity.

Cyclophosphamide (CP) administered ip to pregnant mice on day 10 of gestation (day of plug = day 0) is teratogenic (exencephaly, cleft palate, and limb malformations) at 20 mg/kg and embryolethal at higher doses. In the present study, CP was administered at 1, 5, 10, or 20 mg/kg on day 10 of gestation. Embryos were removed at 8 and 28 hr postdosing, and two embryos from each litter were immediately stained with Nile blue sulfate (NBS) to identify areas of cell death. The remaining embryos were frozen and forelimb buds subsequently removed for flow cytometric (FCM) analysis of the cellular DNA synthetic cycle. Additional litters were examined near term (day 17) for morphological abnormalities; these data were correlated with embryonic toxicity as detected by NBS staining and FCM analysis. Only the highest dose produced malformations. In marked contrast, a dose-related increase in the percentage of limb bud cells in the S (DNA synthetic) phase of the cell cycle was detectable at all doses. Inhibition of DNA synthesis was detected at all doses 8 hr post exposure and persisted through 28 hr for doses greater than or equal to 10 mg/kg. NBS staining indicated increased cell death in the alar plate of the neural tube 28 hr after exposure to 10 mg/kg CP and generally increased cell death in areas of rapid cell proliferation throughout the embryo at 20 mg/kg. The absence of an overt teratogenic response at dose levels that produced significant perturbation of the cell cycle indicates that a measure of embryonic damage can be compensated for or repaired. The implications of these findings for the existence of thresholds in developmental toxicity are discussed.

Retinoic acid-induced spina bifida: evidence for a pathogenetic mechanism.

Treatment of C57Bl/6J mice with three successive doses of all-trans retinoic acid (28 mg kg-1 body weight) on 8 day, 6 h (8d,6h), 8d,12h, and 8d,18h of gestation resulted in a high incidence (79%, 31/39 fetuses) of spina bifida with myeloschisis (spina bifida aperta) in near term fetuses. Twelve hours following the last maternal dose (9d,6h), the caudal aspects of treated embryos, were abnormal, with eversion of the neural plate at the posterior neuropore, as compared to its normal concavity in comparably staged control specimens. This eversion persisted in affected embryos through the time that the posterior neuropore should normally close. The distribution of cell death in control and experimental embryos was determined using vital staining with Nile blue sulphate and with routine histological techniques. Twelve hours following the maternal dosing regimen, experimental embryos showed evidence of excessive cell death, predominantly in the mesenchyme associated with the primitive streak and in the endoderm of the tail gut, both of which are readily identifiable sites of physiological cell death at this stage of development. In addition, the presumptive trunk neural crest cells located in the dorsal midline, cranial to the posterior neuropore, exhibited a marked amount of cell death in the experimental embryos. We propose that the major factor in the generation of spina bifida in this model is excessive cell death in the tail gut and mesenchyme ventral to the neuroepithelium of the posterior neuropore.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of glycoconjugates in the optic vesicle and optic cup.

Lectin histochemical methods and immunohistochemical techniques have been utilized to investigate and partially characterize glycoconjugates in the developing eye. Peanut-lectin-binding sites associated with radial glial cells were found in the diencephalon. In the optic primordia, binding sites associated with radial glia were masked by terminal sialic acid, and only reacted with peanut lectin when pretreated with sialidase. This finding indicates that glycoconjugates associated with diencephalic radial glia contain terminal galactose-beta-(1----3)N-acetyl galactosamine, but glycoconjugates associated with radial glia in the optic primordia contain sialic acid----galactose-beta(1----3)N-acetyl galactosamine. The selective distribution of galactose, N-acetyl galactosamine and fucose associated with radial glial cells has also been demonstrated. We postulate that these distributions mediate the shaping of the developing eye.