Childhood stress, grown-up brain networks: corticolimbic correlates of threat-related early life stress and adult stress response.

BACKGROUND: Exposure to threat-related early life stress (ELS) has been related to vulnerability for stress-related disorders in adulthood, putatively via disrupted corticolimbic circuits involved in stress response and regulation. However, previous research on ELS has not examined both the intrinsic strength and flexibility of corticolimbic circuits, which may be particularly important for adaptive stress responding, or associations between these dimensions of corticolimbic dysfunction and acute stress response in adulthood. METHODS: Seventy unmedicated women varying in history of threat-related ELS completed a functional magnetic resonance imaging scan to evaluate voxelwise static (overall) and dynamic (variability over a series of sliding windows) resting-state functional connectivity (RSFC) of bilateral amygdala. In a separate session and subset of participants (n = 42), measures of salivary cortisol and affect were collected during a social-evaluative stress challenge. RESULTS: Higher severity of threat-related ELS was related to more strongly negative static RSFC between amygdala and left dorsolateral prefrontal cortex (DLPFC), and elevated dynamic RSFC between amygdala and rostral anterior cingulate cortex (rACC). Static amygdala-DLPFC antagonism mediated the relationship between higher severity of threat-related ELS and blunted cortisol response to stress, but increased dynamic amygdala-rACC connectivity weakened this mediated effect and was related to more positive post-stress mood. CONCLUSIONS: Threat-related ELS was associated with RSFC within lateral corticolimbic circuits, which in turn was related to blunted physiological response to acute stress. Notably, increased flexibility between the amygdala and rACC compensated for this static disruption, suggesting that more dynamic medial corticolimbic circuits might be key to restoring healthy stress response.

Osteoblastic cells regulate the haematopoietic stem cell niche.

Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gamma-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.

Thyroid function in Rubinstein-Taybi syndrome.

Rubinstein-Taybi syndrome (RTS) is a genetic syndrome characterized by broad thumbs and halluces, growth retardation, mental retardation, and craniofacial abnormalities. This condition recently was found to be caused by mutations in the gene encoding cAMP response element-binding protein (CREB)-binding protein. As CREB-binding protein has been shown to be a critical coactivator for thyroid hormone receptors, it is plausible that RTS would be characterized by thyroid hormone resistance. In fact, features of RTS, such as mental retardation and short stature, are consistent with thyroid hormone deficiency or resistance. To assess the function of the thyroid axis in RTS, free T4 and TSH were measured in 12 subjects with this syndrome. The free T4 level was normal in all 12 (mean +/- SD, 0.97 +/- 0.20 ng/dL; normal range, 0.73-1.79), as was the TSH level (2.24 +/- 0.87 microU/mL; normal range, 0.3-6.5). Thus, overt thyroid hormone resistance does not appear to be a typical feature of RTS.

Detection of P-glycoprotein in cell lines and leukemic blasts: failure of select monoclonal antibodies to detect clinically significant Pgp levels in primary cells.

Multidrug resistance (MDR) is a salient feature of chemotherapy failure in pediatric patients. One of the most common and well-studied mechanisms implicated in causing MDR is P-glycoprotein (Pgp), an ATP-dependent, transmembrane drug efflux pump. Accurate and reproducible detection of this MDR protein is necessary as it may have important clinical implications. In this study comparing the directly conjugated anti-Pgp monoclonal antibodies UIC2-PE and 15D3-PE to the unconjugated anti-Pgp mAb MRK16, we analyzed cell lines, normal peripheral blood cells, and bone marrow cells from pediatric patients diagnosed with acute myeloid leukemia and acute lymphoblastic leukemia; all samples were also analyzed for Pgp function using rhodamine 123 in order to correlate results from antibody staining with functional activity. For all patient samples evaluated, only MRK16 correlated well with the rhodamine 123 assay. Both the directly conjugated antibodies UIC2-PE and 15D3-PE failed to detect Pgp in almost all cases. Pre-treatment of cells with neuraminidase did not provide a consistent enhancement of antigen detection. Based on these results, we suggest that while UIC2-PE and 15D3-PE may be able to detect the very high levels of Pgp expressing laboratory-cultured cell lines, they are not suitable for clinical application in their currently available conjugated form. When assaying patient samples for Pgp expression and function using flow cytometry, the rhodamine 123 functional assay should be performed in concert with staining with MRK16.

Photogrammetric evaluation in clinical genetics: theoretical considerations and experimental results.

As newer mathematical approaches are applied to the field of clinical genetics accurate methods of craniofacial measurement are increasingly necessary. If photogrammetric techniques are to be used certain theoretical and practical issues must be taken into account. Errors due to projection are particularly important, but systematic and random errors must also be considered. We discuss theoretical aspects of projection errors along with experimental measurements. Systematic errors in excess of 20% were found during simulations of typical clinical conditions, although smaller errors were obtained using techniques practical in a clinical setting. Photogrammetric measurements are potentially valuable in the field of clinical genetics but must be used cautiously.

Functional imaging of brain activity in conscious monkeys responding to sexually arousing cues.

Olfactory cues can elicit intense emotional responses. This study used fMRI in male common marmoset monkeys to identify brain areas associated with sexual arousal in response to odors of ovulating female monkeys. Under light anesthesia, monkeys were secured in a specially designed restrainer and positioned in a 9.4 T magnetic resonance spectrometer. When fully conscious, they were presented with the scents of both ovariectomized and ovulating monkeys. The sexually arousing odors of the ovulating monkeys enhanced signal intensity in the preoptic area and anterior hypothalamus compared to the odors of ovariectomized monkeys. These data corroborate previous findings in monkeys based on invasive electrical lesion and stimulation techniques and demonstrate the feasibility of using non-invasive functional imaging on fully conscious common marmosets to study cue-elicited emotional responses.

Restricted dietary protein in pregnant beef cows: I. The effect on length of gestation and calfhood mortality.

Beef cows fed 0.37 Kg crude protein/day during the last four months of pregnancy had significantly shortened gestation (274 days vs. 282 days) and decreased weight gain (25 Kg vs. 66 Kg) compared to control cows fed 0.96 Kg crude protein/day. Calf mortality in the low protein group was associated with either dystocia (3 calves) or prematurity (2 calves). There were no deaths among control calves. It is suggested that protein malnutrition in late pregnancy may be a contributing factor to neonatal mortality among beef calves.

Immunoglobulin levels in serum and colostral whey or protein-metabolisable energy restricted beef cows.

Aberdeen Angus cows were fed adequate diets or diets restricted in protein and, or metabolisable energy for the last 156 days of gestation to determine effects of nutritional restriction on concentrations of immunoglobulins in serum and colostral whey. There were no significant interactions between the effects of low protein and metabolisable energy on immunoglobulin concentrations. Thus, observed differences in immunoglobulin concentrations between the restricted and adequate dietary groups were attributed to the main effects of treatment. Low protein or metabolisable energy had little overall effect on serum IgM concentrations although levels began to decrease sooner in gestation in restricted animals than in those fed adequate diets. Concentrations of IgG1 in serum of all animals were similar and a precipitous decrease in concentration was noted at about 240 days of gestation and this decrease continued until parturition. Serum IgG2 concentrations increased in all animals as parturition approached. Immunoglobulin concentrations in colostral whey were either similar to or tended to be slightly higher in dietary restricted animals than in animals fed adequate diets although the differences were not significant.

Estimation of repeatability of blood constituents in gestating beef cattle on protein- and energy-restricted diets.

Repeatabilities of blood constituents were calculated for 104 Angus heifers on two separate experiments fed adequate, protein-deficient, energy-deficient, or both protein- and energy-deficient diets. Four statistical methodologies were compared including analysis of variance, principal component (structural) analysis based on the sample covariance and sample correlation matrix, and maximum likelihood. Of 12 blood constituents tested only seven were considered sufficiently important to be included in the analysis. These blood constituents included blood urea nitrogen (BUN), creatinine (Creat), alkaline phosphatase (Alk Phos), total protein (T Prot), total bilirubin (T Bil), cholesterol (Chol) and Iron (Fe). If the standard linear model assumptions were met for heifers on the adequate diet, the estimators appeared to be quite similar for both years except when the correlation coefficient was relatively small. If the assumption of homogeneity of the variance-covariance matrix (compound symmetry) was relaxed, the structural analysis method based on the sample correlation matrix appeared preferable. However, when combining all diets, the maximum likelihood methodology was preferred. Among the specific blood constituents, Alk Phos had the highest repeatability, not only for the heifers on the adequate diet, but also for heifers on other treatments in both years. Repeatabilities for T Prot appeared to be the most consistent over all rations in both years. Repeatability estimates for Fe were high and relative rankings were consistent for both years, while repeatabilities for the other variables were either low and(or) inconsistent.

Effects of prepartum protein restriction in the beef cow on immunoglobin content in blood and colostral whey and subsequent immunoglobin absorption by the neonatal calf.

Protein intake of first-calf beef heifers was restricted during the last 100 days of gestation, and the effects on passive transfer of colostral immunoglobins from the cow to the neonatal calf were examined. There were no significant correlations between concentration of immunoglobins (IgM, IgG1 and IgG2) in the sera or colostrum of the cow and prenatal crude protein consumption (.52 to .98 kg crude protein/day). Absorption of certain colostral immunoglobins (IgG1, and IgG2) by the calf were positively correlated (P less than .01) at 12, 18, 24 and 36 hr after birth to the maternal crude protein consumption. Colostrum was collected from the first milkings of pluriparous dairy cows, and then freeze-dried, mixed and reconstituted to be equivalent to 1 liter of colostrum. Mean IgG1 concentrations for the high and low protein groups were 6.02 +/- .90 and .78 +/- .15 mg . ml-1 (P less than .01), respectively. No relationship (P greater than .05) was found between the concentration of IgM in calf sera and daily crude protein intake of the dam. These data indicate that there was a selective decrease in absorption of IgG1 and IgG2 in calves from heifers fed low protein prenatal diets.

Concentrations of serum constituents in cold-stressed calves from heifers fed inadequate protein and(or) energy.

A study with neonatal calves was conducted to determine the effects of maternal crude protein (CP) and(or) metabolizable energy (ME) malnutrition, cold stress (0 or 21 degrees C), and age on concentrations of selected serum constituents. For each of 2 yr, 60 artificially bred Angus heifers were assigned randomly to a 2 x 2 factorial nutritional plan 150 d before predicted parturition. The diets provided each heifer with either .32 or .96 kg/d of CP and 8.7 or 12.6 Mcal/d of ME. Blood samples were obtained from heifers at parturition and from their calves at birth and at 12, 24, 36, 48, and 72 h of age. Sera were analyzed for concentrations of blood urea nitrogen (BUN), creatinine (Creat), iron, total protein (TProt), alkaline phosphatase (AlkPhos), total bilirubin (TBil), and cholesterol (Chol). Mean correlations of these constituents in calf sera between 12-h adjacency intervals were high, but those between longer times (48 or 60 h) were low. Simple correlations of serum constituents between cows and calves at birth were low except for BUN (r = .578 and .295 for yr 1 and 2, respectively). There were significant main treatment effects for maternal CP consumption on BUN levels, for environmental temperature on BUN, Creat, and TBil levels, and for years on BUN, Creat, iron, and AlkPhos levels in calves. Significant polynomial relationships were found over hours of age for all variables. Blood urea N decreased in normal calves but remained relatively constant at a low level in deficient calves. Year x hour of age interactions occurred for iron, TProt, AlkPhos, TBil, and Chol. Protein x year x hour of age interactions were found for iron and Chol. These results suggest that random sampling times are not useful for decision making during the first 72 h after birth. Consideration must be given to multiple samples taken at specific calf ages, to environmental temperatures, and to maternal protein nutritional levels when interpreting calf blood sera data.

Classification of beef calves as protein-deficient or thermally stressed by discriminant analysis of blood constituents.

Linear discriminant functions hold promise for identifying either protein-deficient or cold-stressed calves based on blood constituents. For each of 2 yr 60 artificially bred Angus heifers were assigned randomly to a 2 x 2 factorial nutritional plan consisting of .32 or .96 kg/d of maternal CP and 8.7 or 12.2 Mcal/d of ME. The calves from these heifers were assigned randomly to environmental chambers set at either 0 or 21 degrees C in a repeated measures design. Linear discriminant functions were computed for 1 yr (training data) and then used to predict the classification of calves for the other year (validation data). Using the original data, the correct classifications of calves to the protein groups were 96, 80, 60, 59, 54, and 51% for blood samples obtained at 0, 12, 24, 36, 48, and 72 h of age, respectively. Using normalized data, corresponding correct classifications to protein groups were 94, 91, 80, 56, 54, and 52%. Results indicate that protein classification should use blood samples obtained within 12 h of age for reasonable success. For cold-stressed calves, correct classifications using original data were 47 (pre-exposure), 72, 54, 70, 67, and 66% for calves at 0, 12, 24, 36, 48, and 72 h of age, respectively. Corresponding correct classifications using normalized data were 54 (pre-exposure), 74, 70, 72, 69, and 77%. Cold stress could be detected after only 12 h of exposure; the time window for testing was much wider than for protein classification, but the classification generally was less discriminative.

Antibody responses in protein-energy restricted beef cows and their cold stressed progeny.

Antibody titers were measured in serum and colostral whey of pregnant beef cows immunized with tetanus toxoid and chicken red blood cells while being fed diets either restricted or nonrestricted in protein and/or metabolizable energy during the last 150 days of gestation. Serum antibody titers were also measured in the colostrum-fed, cold and noncold stressed progeny that were actively immunized with dinitrophenol conjugated to keyhole limpet hemocyanin. In general, there were no major or sustained differences in humoral immune responses to injection of tetanus toxoid or chicken red blood cells between cows fed diets that were adequate or restricted in protein or metabolizable energy. In the few cases where serum antibody titers to tetanus toxoid or chicken red blood cells differed (P less than 0.05) between adequately fed or restricted cows, the differences were no greater than twofold. Anti-chicken red blood cell titers were uniformly low (P less than 0.05) by a magnitude of two to threefold in colostral whey of cows restricted in protein and/or metabolizable energy when compared to titers in cows fed adequate amounts of protein and metabolizable energy. With one exception, neither maternal dietary restriction nor cold exposure had a major effect on the ability of the calves to absorb antitetanus toxoid and chicken red blood cell antibodies from colostrum. The humoral immune responses of all calves to injection of keyhole limpet hemocyanin and dinitrophenol were similar in magnitude.

In vitro migration responses of neutrophils from cows and calves.

The directional (chemotactic) and random migration activities of neutrophils from cows and newborn and 2-week-old calves were determined by use of the chemotaxis-under-agarose assay. Blood samples were stored for 2, 24, or 48 hours and at 4 or 25 C before testing. During the assay, cells were incubated at 17, 27, or 37 C. The assay was found suitable for testing the directional and random migration activities of neutrophils from cattle. Directional migration of neutrophils was diminished (P less than or equal to 0.05) when cells were incubated at 17 or 27 C, compared with data from incubation at 37 C. Random migration of neutrophils was unaffected by test incubation temperature. Significant (P less than or equal to 0.05) differences were found between cows and calves regarding the percentage number and viability and the directional and random migration activities of neutrophils. Neutrophils from cows were adversely affected to a greater extent by prolonged sample storage times or low storage temperature than were neutrophils from calves. Results indicate that a sample storage time of up to 24 hours, a sample storage temperature of 25 C, and a test incubation temperature of 37 C provided optimal conditions for testing the migratory activities of neutrophils from cattle.

Immune responses of the bovine fetus and neonate to Escherichia coli: quantitation and qualitation of the humoral immune response.

The humoral immune responses of fetuses and neonates of Escherichia coli O:26:K60:NM were studied in 26 Angus-Hereford crossbred calves. Bacterin (5.0 X 10(10) organism) was injected in utero directly into the amniotic fluid of seventeen 7- to 8.5-month-old fetuses (principals). Saline solution was injected in the same manner into 9 control fetuses. Colostrum-deprived neonates were allotted to 10 groups and either were euthanatized at birth or were subjected to oral revaccination, challenge inoculation with the homologous organism, or both. The resistance to challenge exposure was a function of previous in utero injection of bacterin, age when challenged, and dose of challenge organisms used. Control calves were susceptible to only a large challenge dose, whereas almost all of the prinicipal calves were resistant. Revaccination of principal calves with bacterin at birth, exposure to the large challenge dose, or both, caused a marked increase in anti-O26 passive hemagglutination titers. Results of quantitative and qualitative radioimunossay indicated that the immune response to the O26 antigen was mainly of the immunoglobulin M (IgM) class, although there were also demonstrable changes in immunoglobulins (Ig) G1 and G2. The actively acquired immune responses were serotype specific, and there was no cross reactivity with 4 other E coli serotypes. An unidentified immunoprecipitate band was observed in immunoelectrophoretograms of whole bovine serum which may represent another class of Ig or which may be a subclass of IgG1 or IgG2.

Immune responses of the bovine fetus and neonate to Escherichia coli: plaque-forming and intestinal immune responses.

The immune responses of 26 Angus-Hereford fetuses and neonates to Escherichia coli O26:K60:NM were studied after bacterin or saline solution was injected (in utero) into the amniotic fluid. Calves were euthanatized at birth or were orally revaccinated; some were challenge exposed with live organisms. The hemolytic plaque assay was used to determine the presence of cells producing immunoglobulins M, G1, and G2 (IgM, IgG1, and IgG2) in 4 segments of the small intestine, mesenteric lymph nodes, and spleen. The passive hemagglutinin activity of intestinal washings was also determined. Anti-O26 passive hemagglutinin activity in the intestinal washings of principal calves was greater than in that of control calves, but in a given segment of the small intestine, usually this activity was relatively small and less consistent than the plaque-forming response. Greater numbers of plaque-forming cells were observed in the small intestine of 14 of the 15 principal calves when compared with the control calves tested.

Regional differences in body temperature in hypothermic and rewarmed young calves.

One- to 7-day-old Holstein bull calves were anesthetized and cold-stressed until their core body temperature (CBT; colonic) was lowered by 10 C. The calves were then rewarmed in warm water, by heat pads or heat lamps, or allowed to recover naturally (unassisted). Temperatures of peripheral tissues, muscles, and the body core were recorded. The time required to lower the CBT of the cold-stressed calves was 168 +/- 11.7 minutes (mean +/- SE). Cold exposure caused a linear decrease in blood, colonic, rectal, and oral temperatures, whereas temperature decreases in the thigh and pectoral muscles, dorsal and ventral thoracic regions, and the hock joint region were generally of greater magnitude and were curvilinear in pattern. By the time the CBT had decreased 1 C, tissue temperatures during cooling were less than (P less than 0.01) the respective temperatures obtained before cooling. The mean time required to rewarm the calves in warm water (47.1 +/- 3.5 minutes) was less than (P less than 0.05) that for the other rewarming methods. The mean rewarming times for the heat pad (128 +/- 12.8) and heat lamp (125.4 +/- 10.9) methods were greater than (P less than 0.05) that for the warm water method, but less than (P less than 0.05) that for the unassisted calves (190.7 +/- 23.1). In general, there was a linear increase in most of the tissue temperatures during recovery although temperatures in the hock joint region were variable. Temperature differences were observed between the thigh and pectoral muscles and between subcutaneous tissues during cooling and recovery. There was poor correlation between the ages of the calves and the time required to decrease their CBT during cooling and also the time required to increase their CBT, regardless of the rewarming method used.

Hematologic values in hypothermic and rewarmed young calves.

Hematologic values were determined in cold-stressed and rewarmed 1- to 7-day-old Holstein bull calves. The animals were anesthetized and then cold-stressed by immersion in water until their core body temperature (colonic) had decreased by 10 C. They were kept at the hypothermic state for an additional 1 hour and then were rewarmed by 1 of 3 external rewarming methods or by natural (unassisted) recovery. Changes observed in the hematologic values of the cold-stressed calves during cooling represented a trend, rather than a direct effect of cold exposure because the values did not differ (P greater than 0.05) from those obtained from the noncold-stressed animals. Nevertheless, a linear decrease (P less than 0.05) in the total number of leukocytes was observed in the cold-stressed calves during cooling when compared with preimmersion values. The leukopenia resulted primarily from a neutropenia (P less than 0.05) and secondarily from decreases in the number of other leukocytes. Minor increases were noticed in the total number of erythrocytes, hemoglobin concentration, and PCV, whereas mean corpuscular values generally remained unchanged during cooling. A rapid and linear increase in the total number of leukocytes was noticed in all cold-stressed calves during recovery. The increase in total leukocytes occurred in all types of leukocytes and particularly in segmented and nonsegmented neutrophils.

Cardiovascular and shivering responses in hypothermic and rewarmed young calves.

Aortic blood pressure, ECG, electromyogram, and heart rate were recorded in cold-stressed and rewarmed young Holstein bull calves. The calves were anesthetized and then cold-stressed by immersion in cold water until their core body temperature (colonic) was lowered 10 C. Hypothermia was continued for 1 additional hour and then the calves were rewarmed by 3 external rewarming methods or were allowed to recover naturally (unassisted). Aortic blood pressure began to decrease in cold-stressed calves by the time their core body temperature had decreased 2 C and continued to decrease during cooling. Heart rate initially increased then decreased with cooling. Blunting of the systolic blood pressure peaks and appearance of extraneous waveforms that obscured the normal component waveforms of the ECG complex were also observed during cooling. Aortic blood pressure and heart rate of cold-stressed calves increased soon after the start of recovery and eventually returned to base line even though the rate of recovery varied depending on the method of rewarming. The component waveforms of the ECG complex became more discernible as rewarming of the cold-stressed calves progressed.

Serum chemical values in hypothermic and rewarmed young calves.

Serum chemical values were determined in cold-stressed Holstein bull calves ranging from 1 to 7 days of age. The animals were anesthetized and cold-stressed until their core body temperature (colonic) was lowered 10 C. Animals were then rewarmed in warm water, with heat pads or heat lamps, or were allowed to recover naturally (unassisted) at room temperature. Blood samples were collected at selected intervals during cooling and recovery. Increases (P less than 0.05) were observed in the concentrations of glucose, calcium, phosphorus, iron, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, total protein, albumin, total globulin, serum urea nitrogen, uric acid, total bilirubin, indirect bilirubin, and cholesterol in the cold-stressed calves during cooling. Concentrations of chloride and insulin decreased (P less than 0.05) during the same period. Changes observed in many of the serum chemical values during rewarming were generally the reverse of the respective changes that occurred during cooling, although insulin values became exceedingly high in some cases midway or near the end of recovery. Serum enzyme values also remained high during most of recovery. Data did not indicate a clear advantage of one method of rewarming over the other methods used in terms of return of the serum chemical values to normal.