The importation of hematogenous precursors by the thymus is a gated phenomenon in normal adult mice.

Hematogenous precursors repopulate the thymus of normal adult mice, but it is not known whether this process is continuous or intermittent. Here, two approaches were used to demonstrate that the importation of prothymocytes in adult life is a gated phenomenon. In the first, age-dependent receptivity to thymic chimerism was studied in nonirradiated Ly 5 congenic mice by quantitative intrathymic and intravenous bone marrow (BM) adoptive transfer assays. In the second, the kinetics of importation of blood-borne prothymocytes was determined by timed separation of parabiotic mice. The results showed that >60% of 3-18-wk-old mice developed thymic chimerism after intrathymic injection of BM cells, and that the levels of chimerism (range, 5-90% donor-origin cells) varied cyclically (periodicity, 3 to 5 wk). In contrast, only 11-14% of intravenously injected recipients became chimeric, and chimerism occurred intermittently (receptive period approximately 1 wk; refractory period approximately 3 wk). In the intravenously injected mice, chimerism occurred simultaneously in both thymic lobes; gate opening occurred only after most intrathymic niches for prothymocytes had emptied; and the ensuing wave of thymocytopoiesis encompassed two periods of gating. These kinetics were confirmed in parabiotic mice, and in cohorts of mice in whom gating was synchronized by an initial intrathymic injection of BM cells. In addition, a protocol was developed by which sequential intravenous injections of BM cells over a 3 to 4 wk period routinely induces thymic chimerism in the apparent absence of stem cell chimerism. Hence, the results not only provide a new paradigm for the regulation of prothymocyte importation during adult life, but may also have applied implications for the selective induction of thymocytopoiesis in nonmyeloablated hosts.

Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins.

We have previously demonstrated that astrocytes synthesize and secrete apolipoprotein E in situ. In the present work, primary cultures of rat brain astrocytes were used to study apolipoprotein E synthesis, secretion, and metabolism in vitro. The astrocytes in culture contained immunoreactive apolipoprotein E in the area of the Golgi apparatus. Incubation of the astrocytes with [35S]methionine resulted in the secretion of labeled immunoprecipitable apolipoprotein E, which constituted 1-3% of the total secreted proteins. The apolipoprotein E secreted in culture and the apolipoprotein E in rat brain extracts differed from serum apolipoprotein E in two respects: both had a slightly higher apparent molecular weight (approx. 36,000) and more acidic isoforms than serum apolipoprotein E. Sialylation of the newly secreted apolipoprotein accounted for the difference in both the apparent molecular weight and isoelectric focusing pattern of newly secreted apolipoprotein E and plasma apolipoprotein E. The astrocytes possessed apolipoprotein B,E(LDL) receptors capable of binding and internalizing apolipoprotein E-containing lipoproteins. The uptake of lipoproteins by the cells led to a reduction in the number of cell surface receptors and to the intracellular accumulation of cholesteryl esters. Since apolipoprotein E is present within the brain, and since brain cells can express apolipoprotein B,E(LDL) receptors, apolipoprotein E-containing lipoproteins may function to redistribute lipid and regulate cholesterol homeostasis within the brain.

Screening for adult phenylketonuria in state psychiatric hospitals.

The authors report the results of screening patients admitted to a state psychiatric hospital for unrecognized adult phenylketonuria. The results suggest that unrecognized adult phenylketonuria is very uncommon in these patients.

The effects of photoperiod and lid suture on eye growth in chickens.

Three models of postnatal eye enlargement in leghorn chicks (surgical fusion of the eyelids, MES; exposure to continuous light, 24L; prolonged darkness, OL) were characterized on a morphologic and temporal basis, and a relationship between photoperiod and eye growth was described. While pronounced enlargement of the eye was evident in the OL and MES groups after 6 wk, a 15-wk experimental period was necessary to produce significant (P less than 0.05) eye enlargement under 24L. This enlargement was unaffected by pinealectomy and was characterized by increased equatorial diameter and decreased axial length. The macrophthalmos resulting from both MES and OL was characterized by increases in absolute and relative eye weights, axial length, and equatorial diameter. The MES eyes, however, showed a pronounced bulging of the cornea and increased anterior chamber depth and equatorial diameter, while those from the OL group had a flattened cornea and decreased anterior chamber depth. Finally, a relationship between photoperiod and eye growth was established and was best described by an inverse, continuous semilogarithmic function.

Effects of sentence constraint on priming in natural language comprehension.

In 4 cross-modal naming experiments, researchers investigated the role of sentence constraint in natural language comprehension. On the sentence constraint account, incoming linguistic material activates semantic features that in turn pre-activate likely upcoming words. The 1st and 2nd experiments investigated whether stimulus offset asynchrony played a critical role in previous studies supporting the sentence constraint account. The 3rd and 4th experiments examined further predictions of the sentence constraint account, in particular whether pre-activated words would compete for activation. In Experiment 3, the researchers manipulated whether an expected target word had a close competitor and found that response to the expected word was facilitated regardless of the proximity of a competitor. The 4th experiment established that close competitors were primed by the sentence frames and should have been available to compete with expected target words. Thus, word-level representations did not compete for activation.

Molecular cloning and mRNA expression of porcine interleukin-12.

Interleukin (IL)-12 is a heterodimeric cytokine consisting of 35 and 40 kDa subunits, produced primarily by phagocytic cells in response to bacteria or bacterial products. IL-12 is important in the regulation of both innate and antigen-specific immunity through its stimulatory effects on NK cells and cytotoxic lymphocytes. Reverse transcriptase-polymerase chain reaction with primers derived from human sequence was used to clone the p35 and p40 subunits of porcine IL-12. Predicted amino acid sequences for both subunits are approximately 85% homologous to their human cognates but contain a 3aa addition and a 4aa deletion in p35 and p40 subunits, respectively. The high degree of similarity indicates the proteins may be cross reactive, an important consideration in pig-human xenotransplantation. Both subunits of pIL-12 are constitutively expressed in a variety of porcine tissues. Highest levels of the p40 subunit were found in lymphoid tissues including inguinal and mesenteric lymph nodes, Peyer's patches, spleen and thymus. The p35 subunit was also detected in these tissues. Levels of mRNA encoding the p40 subunit, but not the p35 subunit, were rapidly increased in alveolar macrophages stimulated with lipopolysaccharide or killed Staphylococcus aureus. Thus, the heterodimeric subunits appear to be differentially regulated at the transcriptional level. Since p40 also self-associates to form inactive homodimers, differential expression may be a mechanism for regulating IL-12 activity.

Bacterially induced activation of interleukin-18 in porcine intestinal mucosa.

Interleukin (IL)-18 is a cytokine with structural and functional properties similar to IL-1beta and IL-12, respectively. It is activated by caspase-1 cleavage, like IL-1beta, and induces interferon (IFN)-gamma, like IL-12. In order to study the role of IL-18 in the immune response to infectious diseases of mucosal surfaces we cloned and expressed porcine IL-18 and developed antibodies to the protein. Porcine IL-18 retains the caspase-1 cleavage site present in other mammalian IL-18 proteins, but has two potential N-linked glycosylation sites not found in those proteins. Porcine interleukin-18 mRNA and protein are expressed in immune tissues including lymph nodes and gut associated lymphoid tissues. Specific cell types containing IL-18 include lung and splenic macrophages, nonadherent spleen cells and intestinal epithelial cells. Although IL-18 transcription is moderately induced by lipopolysaccharide, the magnitude and total expression level are small compared to those of interleukin-1beta. In vivo and ex vivo infection of intestinal mucosa with Salmonella choleraesuis resulted in a decrease in size of IL-18, consistent with cleavage of the preprotein by caspase-1. Thus, IL-18 is present in mucosal tissues where it could play a role in the immune response to invading pathogens.

Construction of internal cDNA competitors for measuring IL-10 and IL-12 cytokine gene expression in swine.

A competitive PCR assay (cPCR) was used to quantify swine cytokine responses to parasite infection. Internal standards (deleted cDNA competitor molecules [DcDNA mimics]) were produced and tested for swine interleukin-12 (IL-12), interleukin-10 (IL-10) and hypoxanthine phosphoribosyltransferase (HPRT) from PCR generated cDNA cloned in plasmid vectors. Deletion clones for the cDNA competitor molecules (DcDNA mimics) were generated for IL-10, IL-12 and HPRT by PCR in a single step and verified by (1) amplification of the expected smaller PCR product with the original primers, (2) appropriate fragment size released by restriction digestion of the deleted clone, and (3) correct sequence of the new DcDNA insert. DcDNA mimics were used to quantitate cytokine gene mRNA production during experimental and natural infections of swine with the gastrointestinal nematode parasite Trichuris suis. Mesenteric lymph node cells were collected from control and infected pigs at the time of maximal pathogenicity (35 days after infection) and snap frozen. After RNA extraction, samples were reverse transcribed (RT) to cDNA. cPCR was performed using the housekeeping gene HPRT DcDNA mimic and HPRT specific primers to insure RNA integrity and concentration. Cytokine cDNA content in these samples was then quantitated using cytokine mimics and gene specific primers. IL-10 gene expression in MLN draining the colon of pigs experimentally infected with T. suis increased 10-20 fold at day 35 compared to control pigs. IL-12 gene expression was not detectable in MLN of these pigs, but was detectable in MLN of pigs exposed naturally to T. suis on a contaminated dirt lot that also exhibited signs of secondary bacterial invasion. Swine IL-10 and IL-12 gene expression can be quantitated in local mesenteric tissues. This cPCR assay will enable scientists to quantitate cytokine gene expression in swine and determine the nature of immune responses to important infectious diseases.

Mechanisms of vaccine adjuvanticity at mucosal surfaces.

The vast majority of pathogens invade via mucosal surfaces, including those of the intestine. Vaccination directly on these surfaces may induce local protective immunity and prevent infection and disease. Although vaccine delivery to the gut mucosa is fraught with obstacles, immunization can be enhanced using adjuvants with properties specific to intestinal immunity. In this review, we present three general mechanisms of vaccine adjuvant function as originally described by Freund, and we discuss these principles with respect to intestinal adjuvants in general and to the prototypical mucosal adjuvant, cholera toxin. The key property of intestinal adjuvants is to induce an immunogenic context for the presentation of the vaccine antigen. The success of oral vaccine adjuvants is determined by their ability to induce a controlled inflammatory response in the gut-associated lymphoid tissues, characterized by the expression of various costimulatory molecules and cytokines. An understanding of the specific molecular mechanisms of adjuvanticity in the gut will allow the rational development of safe and effective oral vaccines.

Application of a polymerase chain reaction assay for detection of proliferative enteritis-affected swine herds.

A polymerase chain reaction (PCR) assay was used to confirm the presence of ileal symbiont (IS) intracellularis in 3 swine herds with a history of proliferative enteritis (PE). Two pooled fecal specimens, each comprising 5 individual stool samples, were collected from pen floors to screen for the presence of IS intracellularis and determine the age range of pigs shedding the organism. IS intracellularis was detected in the feces of clinically normal 10-25-week-old grower/finisher pigs, indicating that this age range of pigs was the main source of infection for younger nursery pigs. Shedding continued without clinical disease when 10-100 g/ton of tylosin or 10 g/ton of chlortetracycline was added to the feed. PCR testing of pooled fecal samples can be used to identify groups of pigs affected with PE. The results of this study indicate that this PCR assay has the potential to accurately assess the IS intracellularis infection status of swine herds and the association of IS intracellularis with PE and growth performance.

Pathophysiologic correlates of acute porcine pleuropneumonia.

OBJECTIVE: To develop and evaluate an in vivo model to study early events in the pathogenesis of acute porcine pleuropneumonia. ANIMALS: Thirty-six 6- to 8-week-old pigs. PROCEDURE: Pigs were inoculated intranasally or endotracheally with Actinobacillus pleuropneumoniae; inoculation routes were compared by evaluation of clinical signs, gross and microscopic lung lesions, hematologic changes, serum zinc, iron, and haptoglobin concentrations, and inflammatory cytokines. RESULTS: The 2 inoculation routes resulted in similar findings, although intranasal inoculation caused unilateral gross lung lesions, whereas endotracheal inoculation caused bilateral gross lesions. Clinical signs of disease were observed < 2 hours after endotracheal inoculation and 6 to 8 hours after intranasal inoculation. Total WBC counts did not differ significantly after inoculation by either inoculation route, although band neutrophils increased significantly. The earliest findings associated with A pleuropneumoniae inoculation, irrespective of route, were decreased serum zinc and iron concentrations. Serum haptoglobin concentrations were significantly increased after inoculation. Inoculation induced rapid influx of macrophages into the lung and local induction of proinflammatory cytokines. Northern blot analysis of total RNA from lung tissue indicated that inoculated pigs had increased concentrations of interleukin (IL)-1beta, IL-1alpha, and IL-8; tumor necrosis factor messenger RNA concentration was not increased. CONCLUSIONS: Endotracheal inoculation with A pleuropneumoniae rapidly and consistently induced diffuse bilateral pneumonia; thus, this method may be useful for the study of acute pathophysiologic changes associated with bacterial pneumonia and may provide an experimental model for testing modalities for prevention and treatment of this and other respiratory tract diseases of pigs.

Dietary phosphorus levels during growth of brown egg type replacement pullets.

Caged Sex-Sal (DeKalb Warren) replacement pullets were fed diets containing .30%, .35%, or .41% available phosphorus from 0 to 20 weeks of age; in a second study pullets were fed the above levels plus a level of .25% available phosphorus from 2 to 20 weeks of age. Some of the pullets were fed diets restricted by 11 to 16% from 8 weeks of age. Reducing the dietary phosphorus did no harm weight gain, feed intake, feed conversion efficiency, bone ash or total calcium and inorganic phosphorus levels in plasma. There was a very small but significant reduction in weight gain and feed intake when .30% or .35% available phosphorus was fed from 0 to 4 weeks of age, but this difference disappeared at the later ages. With the nonstimulatory lighting schedule used, plasma phosphorus decreased markedly in the latter phase of the studies at all levels of dietary phosphorus and thus represented a nondietary age effect. These results show that dietary available phosphorus for cage, brown egg type pullets on full or restricted feeding programs can be decreased to a level as low as .25% from 2 to 20 weeks, and .30% from hatching to 20 weeks without adverse effect.

Effect of selected light treatments on pineal weight and lipid content in the cockerel (Gallus domesticus).

The effect of early and prolonged exposure of growing cockerels to selected photoperiods and wavelengths of light on pineal gland weight and lipid composition was investigated. The six treatments included 14L:10D white (control), 24L:OD white, OL:24D dark, and 14L:10D narrow-band blue, green or red light. Pineal gland fresh and dry weights were greatest under the white light treatment and least under the dark and red treatments. Examination of histological preparations and biochemical analysis both indicated that the absence of light depleted pineal lipid. When expressed as a percent of dry tissue weight, lipid from the colored light treatments was significantly greater than from the control. We conclude that both the photoperiod and the wavelength of light are capable of influencing pineal gland lipid metabolism in the cockerel.

Pineal gland lipid characterization in the seven-week-old cockerel (Gallus domesticus).

The composition of pineal gland lipid was characterized in young cockerels. The total pineal lipid was 159.7 microgram, which was equivalent to 3.0 and 14.1% of fresh and dry tissue weight, respectively. The major phospholipids were phosphatidyl choline (63.1%), phosphatidyl ethanolamine (16.1%), and sphingomyelin (14.7%). Palmitic, stearic, oleic, and arachidonic acids composed 27.9%, 19.4%, 15.8%, and 13.7% of the phospholipid fatty acid fraction, respectively. Free fatty acids (38.4%), cholesterol (17.6%), and cholesterol ester (17.2%) comprised the major fraction of neutral lipids, while the predominant neutral lipid fatty acids were palmitic (26.0%) and oleic (23.8%). The percentage of wet tissue weight that is lipid in the pineal is considerably less when compared to brain cortex or retina. When chickens are compared to several mammalian species, there is little difference in percent lipid per milligram pineal tissue, but definite differences exist in the percentage of various lipid classes.

Pinealectomy and light environment effects on testicular and comb development in the 46-day-old broiler cockerel.

Four replicate experiments were conducted to determine the effects of pinealectomy and environmental lighting on testes and comb weights in the broiler cockerel. Birds were housed in brooder batteries under a 14L:10D fluorescent white light regime for 2 weeks and then allotted to light-controlled chambers. Surgery was performed when the chicks were 3 to 5 days old. The fluorescent light treatments were 14L:10D green (narrow-band, 545 nm peak), 14L:10D cool white, and constant darkness. At the end of the 46-day experimental period, testes weights were determined and comb weight recorded. Pinealectomy did not affect testes weights or comb development. Darkness significantly (P less than .05) depressed testes and comb weight. This suggests that lack of light, but not pinealectomy, affects circulating hormone levels and/or tissue responsiveness in the young cockerel.

Effect of herring oil on body weight, comb size and gonadal development in the chick.

A diet with 20% herring oil (0.55% linoleate) gave comparable growth responses to a diet containing 20% corn oil (12.07% linoleate) in 4-week-old male and female White Leghorn chicks. No additive or synergistic growth responses were noted in female chicks fed a mixture of herring oil and corn oil. Decreased testes size and changes in testicular histology occurred in young cockerels fed herring oil. Comb responses were dissimilar in male and female chicks fed fish oil.

Description and surgical removal of the cranial cervical ganglia in 3- to 5-day-old chicks.

The cranial cervical sympathetic ganglion (CCG) provides the primary innervation of the pineal gland in several gallinaceous species. The CCG is located at the base of the skull near the exoccipital bone, dorsal to the level of the vagus and glossopharyngeal nerves. It occupies a much larger volume and appears pinkish-gray, instead of white, when compared to the petrosal ganglion. To surgically remove the CCG, chicks were anesthetized with halothane vapor. Following a small skin incision, blunt dissection was used to expose the CCG lying adjacent to the internal carotid. The ganglion was grasped with small forceps and pinched free of its fine neural connections to adjacent nerves. The success of the surgery was confirmed visually and by complete adrenergic dennervation of the pineal gland. The entire surgical procedure required approximately 20 min per bird. Mortality was less than 20% overall.