Quantitative and qualitative analysis of stability for 16 serum immunoregulators over 50 freeze-thaw cycles.

OBJECTIVES: To evaluate the reliability of data from the assay of bio-archived specimens, a 50-freeze-thaw-cycle (FTC) degradation study of fresh sera was conducted to test the stability of 16 immunoregulators. METHODS: Twenty de-identified serum specimens were obtained from volunteers at United Health Services-Wilson Memorial Hospital. Specimens were stored at -20°C and underwent daily 1 h thawing and subsequent freezing for each FTC over 50 consecutive days. Immunoregulator concentrations were assessed via enzyme-linked immunosorbent assay (ELISA) in participant samples at 2 FTC (baseline), 25 FTC, and 50 FTC. Specific immunoregulators observed in the study were C-reactive protein (CRP), interleukin (IL)-1α, 4, 6, 8, 10, monocyte chemoattractant protein-1 (MCP-1, CCL2), monocyte chemoattractant protein-2 (MCP-2, CCL8), eotaxin-1, thymus-and-activation-regulated chemokine (TARC, CCL17), regulated on activation normal T-cell expressed and secreted (RANTES, CCL5), growth-regulated oncogene-alpha (GRO-α, CXCL1), small inducible cytokine A1 (I-309, CCL1), interferon-gamma (IFN-γ), interferon-gamma inducible protein-10 (IP-10, CXCL10), and tumor necrosis factor-alpha (TNF-α). RESULTS: Quantitative stability of serum immunoregulators: Serum CRP, IL-8, IL-10, IFN-γ, IP-10, and eotaxin-1 levels appear to be statistically equivalent from baseline to 50 FTC (p ≤ .05). Retention of patterns in serum immunoregulators: patterns across FTC were retained for TARC (age) and CRP, IFN-γ, and MCP-2 (sex). CONCLUSIONS: While the effect of multiple FTC on serum immunoregulator levels may not replicate prolonged freezer storage, the results of this study provide valuable information on the robustness of immunoregulators for research using bio-archived sera.

Establishing the structure and replicability of personality profiles using the HEXACO-PI-R.

Previous attempts to identify personality profiles in the five-factor and HEXACO models of personality have produced inconsistent results. Here, using data from four independent samples, each with approximately 90,000 international respondents to the 100-item HEXACO Personality Inventory-Revised (HEXACO-PI-R), we demonstrated that a five-profile solution fit the data well. Exploratory analyses suggested that this solution was also consistent across gender and age groups. The five-profile structure replicated well with larger subsamples, but could not be reproduced consistently with samples of fewer than 500 individuals. However, even with small samples, the five-profile structure could be applied using the parameters obtained with the larger samples. We used HEXACO theory along with agency-communion and attachment theories to offer preliminary explanations and labels for the five profiles. We discuss how these theories, combined with parameter estimates provided by our research, can be used to generate and test hypotheses to validate the five-profile structure and evaluate its utility for personality research and other applications.

Intranasal inoculation of recombinant DNA vaccine ABA392 against haemorrhagic septicaemia disease.

We evaluate the efficacy of recombinant DNA vaccine ABA392 against haemorrhagic septicaemia infection through intranasal administration route by targeting the mucosal immunity. The DNA vaccine was constructed and subjected to animal study using the Sprague Dawley (SD) rat. The study was divided into two major parts: (i) active and (ii) passive immunization studies, involving 30 animals for each part. Each group was then divided into five test groups: two test samples G1 and G2 with 50 and 100 µg ml-1 purified DNA vaccine; one positive control G5 with 106  CFU per ml formalin-killed PMB2; and two negative controls, G3 and G4 with normal saline and pVAX1 vector. Both studies were conducted for the determination of immunogenicity by total white blood cell count (TWBC), indirect ELISA and histopathological changes for the presence of the bronchus-associated lymphoid tissue (BALT). Our findings demonstrate that TWBC, IgA and IgG increased after each of the three vaccination regimes: groups G1, G2 and G5. Test samples G1 and G2 showed significant differences (P < 0·05) compared to the negative controls, G3 and G4, but no significant differences from the positive control G5. Groups G1, G2 and G5 showed more formation of BALT compared to the negative controls, G3 and G4. Our results show that intranasal inoculation of recombinant DNA vaccine ABA392 can provoke mucosal immunity which makes it a potential prophylactic against HS. SIGNIFICANCE AND IMPACT OF THE STUDY: New approach of combating haemorrhagic septicaemia disease among bovines by recombinant DNA vaccine is crucial to overcome the loss of edible products from the infected bovines. DNA vaccine can potentially serve as a better immunogen which would elicit both cellular and humoral immunity, and it is also stable for its molecular reproduction. This research report demonstrates an effective yet simple way of administering the DNA vaccine via the intranasal route in rats, to provoke the mucosal immunity through the development of immunoglobulins IgA, IgG and bronchus-associated lymphoid tissue which guard as the first-line defence at the host's mucosal lining.

Content specificity of attentional bias to threat in post-traumatic stress disorder.

BACKGROUND: Attentional bias to affective information and reduced cognitive control may maintain the symptoms of post-traumatic stress disorder (PTSD) and impair cognitive functioning. However, the role of content specificity of affective stimuli (e.g., trauma-related, emotional trauma-unrelated) in the observed attentional bias and cognitive control is less clear, as this has not been tested simultaneously before. Therefore, we examined the content specificity of attentional bias to threat in PTSD. METHODS: PTSD participants (survivors of a multistory factory collapse, n=30) and matched controls (n=30) performed an Eriksen Flanker task. They identified the direction of a centrally presented target arrow, which was flanked by several task-irrelevant distractor arrows pointed to the same (congruent) or opposite direction (incongruent). Additionally, participants were presented with a picture of a face (neutral, emotional) or building (neutral=normal, emotional=collapsed multistory factory) as a task-irrelevant background image. RESULTS: We found that PTSD participants produced overall larger conflict effects and longer reaction times (RT) to emotional than to neutral stimuli relative to their healthy counterparts. Moreover, PTSD, but not healthy participants showed a stimulus specific dissociation in processing emotional stimuli. Emotional faces elicited longer RTs compared to neutral faces, while emotional buildings elicited faster responses, compared to neutral buildings. CONCLUSIONS: PTSD patients show a content-sensitive attentional bias to emotional information and impaired cognitive control.

Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML.

Therapy directed against oncogenic FLT3 has been shown to induce response in patients with acute myeloid leukemia (AML), but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant AML cell lines by sustained treatment with the FLT3 inhibitor sorafenib. Parental cell lines carry the FLT3-ITD (tandem duplication) mutation and are highly responsive to FLT3 inhibitors, whereas resistant cell lines display resistance to multiple FLT3 inhibitors. Sanger sequencing and protein mass-spectrometry did not identify any acquired mutations in FLT3 in the resistant cells. Moreover, sorafenib treatment effectively blocked FLT3 activation in resistant cells, whereas it was unable to block colony formation or cell survival, suggesting that the resistant cells are no longer FLT3 dependent. Gene expression analysis of sensitive and resistant cell lines, as well as of blasts from patients with sorafenib-resistant AML, suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype, which was further supported by next-generation sequencing and phospho-specific-antibody array analysis. Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in resistant cell lines. Gedatolisib significantly extended survival of mice in a sorafenib-resistant AML patient-derived xenograft model. Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD-dependent AML results in resistance to drugs targeting FLT3.

Effect of maternal oral hydration therapy in oligohydramnios.

This was a randomized controlled trial on 64 pregnant women from 32 to 35 weeks gestation, in one year period to determine the effect of maternal hydration by oral water in oligohydramnios amniotic fluid index (AFI) ≤ 5. Studied women were randomly divided into two groups. Group A (intervention group) women were instructed to drink 2 liters of water within 2 hours and from the next day extra 2 liters of water daily for 7 days. Group B (control group) women were allowed for routine water intake. AFI was done after 2 hours, 24 hours and 7 days of oral hydration therapy in both the groups. P values less than 0.05 was considered statistically significant. Pre-treatment mean AFI was 4.77 ± 0.42 (mean ± SD) vs. 4.80 ± 0.43 (mean ± SD) and post treatment AFI after 2 hours was 6.35 ± 0.65 vs. 4.81 ± 0.42; after 7 days was 7.08 ± 0.21 vs. 5.0 ± 0.20 in oral hydration group and control group respectively. Delivery at 37-40 weeks was 53.1% vs. 12.4%, normal vaginal delivery in 71% vs. 21.8%, caesarean section in 29% vs. 78.2% and low birth weight babies were 12.5% vs. 81.25% in intervention and control group respectively. Foetal outcome was healthy in 87.1% vs. 59.4%, asphyxiated in 12.9% vs. 50% and perinatal death was 3.22% vs. 21.8% between intervention and control group. Still born were 6.3% cases in control group. Maternal oral hydration therapy significantly increases the AFI, reduces the caesarean section rate and improves the foetal outcome.

Verrucous carcinoma of vulva.

Vulval carcinoma is an uncommon disease of old women and may manifest as verrucous carcinoma. Intraepithelial cancer of the vulna of women in their twenties and thirties is increasing. Invasive carcinoma of vulva usually appears long time after intraepithelial neoplasia or chronic irritation. We are presenting one young lady of 21 years who noticed whitening of vulva since her childhood and irregular vulval itching in that area. After nine to ten years she further noticed swelling in that area which used to discharge off an on along with itching. As she got married and had disturbed sexual life, on consulting with a specialist doctor, the lesion was diagnosed as an ulcerated malignant growth. Further investigation revealed the lesion to be as verrucous carcinoma of vulva. This is the first report of such case in our country. Any persistent complaints of vulva like itching, discharge, even ulcer should be taken care of for detection of intraepithelial vulvar cancer.

The relationship of placental weight with birth weight.

To assess the relationship between placental weight and birth weight, two hundred forty six pregnant mothers, who were otherwise healthy, were prospectively followed in a city hospital during antenatal period until delivery and immediate post-partum period. Height of mothers was measured initially and weight measured at each visit during the antenatal check-up. Placental weight and birth weight of babies were measured by one of the authors immediately after delivery by a weighing scale. Eighty one percent of the mothers were between the age group of 20-29 years. The BMI of 92% mothers was 18.5 and above. Most of the mothers came both with primigravida (42%) or second gravida (33%) and in 25% cases 3rd or onwards. In 49% cases the placental weight was between 401-500 gm, in 30% cases >500 gm and in 21% cases 400 gm or less. There was delivery of appropriate-birth-weight babies in 85% cases and low-birth-weight babies in 15% cases. It was observed that a very strong correlation existed between placental weight and birth weight (r = 0.391, p<0.001). Even this correlation was stronger in small for gestational age babies. However, there was no correlation between placental weight and APGAR score at one minute. It is concluded that increment of birth weight occurs with increase of placental weight. If placental weight can be measured by ultrasonography in second or early third trimester of pregnancy birth weight is possible to be assessed and appropriate measure can be taken to increase the birth weight.

Protein kinase C activation promotes microtubule advance in neuronal growth cones by increasing average microtubule growth lifetimes.

We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow-a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance.

Capture of rare cells in suspension with antibody-coated polystyrene beads.

A method for the separation of one cell type present in small number from a predominant mixture of cell types using macroscopic polystyrene beads is demonstrated. An antibody specific to murine leukocytes (CD45) was adsorbed to the surface of the beads. Beads and murine hybridoma B cells were placed in test tubes and periodically inverted at fixed time intervals, causing the beads to settle through the suspension under creeping flow conditions. Capture was dependent upon interception: the captured cells must have traveled along streamlines that brought them to within a cell radius of the bead surface. B cells attached to 99-micrometer beads (maximum shear rate 8.1 s-1) were captured with greater efficiency but in lesser quantity than those attached to 170-micrometer beads (maximum shear rate 13.9 s-1). Cell capture unexpectedly reached a plateau in less than 2 h, a phenomenon that appears to involve changes in both the cells and the beads. Capture of cells was effective out to dilutions of 1:10 000 with purity in the captured population of better than 74%. This method allows for the study of physical parameters important for cell attachment and capture as well as for practical separation of rare cells.

Immunocytochemical detection and spatial distribution of myosin light-chain kinase in preimplantation mouse embryos.

As a follow-up to our previous study on the role of myosin light-chain kinase (MLCK), a Ca2+/calmodulin-dependent enzyme, in the development of preimplantation mouse embryos, we examined the presence and pattern of distribution of MLCK during preimplantation development of the mouse by whole-mount, indirect immunocytochemistry and by Western blotting, using a monoclonal antibody against MLCK. At all stages of preimplantation development, the nucleus was brightly stained with an unstained region around the nucleus, and regions near the cell membrane were also brightly stained. Using the optical sectioning capability of the confocal laser scanning microscope, we found that, up to the eight-cell stage, the regions of cell contact were mostly unstained, but along with the process of compaction, cell contact regions showed a clear staining pattern along with clearing of the cytoplasm. During formation of the blastocyst, a ring of immunofluorescence was found at the margin of the blastocoel. In the blastocyst, cells of the inner cell mass were less immunofluorescent than trophectoderm cells. These staining results appear to be due to specific immunoreaction between MLCK and the antibody, because the staining patterns were abolished when the antibody was preabsorbed by MLCK purified from chicken gizzard smooth muscle. In Western blotting of blastocysts, we found a band at 130 kD. We also show by immunoblotting and immunohistochemistry of various mouse tissues that the antibody used in this study has cross-reactivity to MLCK of various muscle and non-muscle tissues of the mouse. The presence and spatial distribution of MLCK at various stages of preimplantation development of the mouse suggest that it could play a crucial role in the regulation of the contractile events involved in the initial differentiation that occurs during formation of the mouse blastocyst.

Regulation of preimplantation development of mouse embryos: effects of inhibition of myosin light-chain kinase, a Ca2+/calmodulin-dependent enzyme.

We have examined the effects of ML-9 and wortmannin, which are, respectively, specific reversible and irreversible inhibitors of myosin light-chain kinase, a Ca2+/calmodulin-dependent enzyme, on preimplantation development of the mouse in an attempt to establish a regulatory role for this enzyme in preimplantation development. When late two-cell stage embryos were treated continuously with ML-9 or wortmannin at a concentration of 0, 1, 5, 10, or 15 microM, compaction and formation of the blastocyst were inhibited in a dose-dependent manner. Stage-specific treatment with ML-9 at 25 microM induced stage-specific responses of embryos after the eight-cell stage during the processes of compaction and cavitation. These morphological responses included aborted compaction, decompaction of compacted embryos, and the inability of embryos to form a cavity. These morphological effects were reversible, but, since cell proliferation was inhibited, the "recovered" embryos were small. Counting of cells on day 4 of culture, in both continuously treated and stage-specifically treated embryos, showed that the effect of ML-9 on cell proliferation was also dose-dependent. Wortmannin also had stage-specific effects at 15 microM, but these effects were irreversible and were more deleterious than those of ML-9. With neither inhibitor was there any apparent effect at the two-cell or the four-cell stage, although wortmannin inhibited cell division when applied stage-specifically at the four-cell stage. These results indicate that myosin light-chain kinase may be an important enzyme in the first steps of differentiation and in the maintenance of the differentiated state during preimplantation development of the mouse.

Body composition and fat distribution during the first 2 weeks of gestation in ad lib.-fed and energy-restricted rats.

Knowledge about changes in body composition during gestation is of interest when estimating energy requirements during pregnancy, and relevant since reproduction is often surprisingly well maintained in malnourished females. Due to difficulties in conducting such studies in humans, studies in rats are of interest. Therefore, maternal retention of fat and fat-free weight was estimated in rats during the first 2 weeks of gestation and during a corresponding time period in virgin controls. Groups fed ad lib. or 70% of ad lib. intake during the 4 weeks preceding conception and during gestation were studied. Retention was estimated by comparing pregnant and virgin rats with rats killed at the time of conception. Body fat was analysed chemically and fat-free weight was body weight minus body fat. Each rat was divided into twelve to fourteen parts and the fat content of each part was analysed. Pregnant ad lib.-fed rats retained more fat and fat-free weight than did virgin ad lib.-fed controls. In the energy-restricted group the pregnant rats retained more fat-free weight while virgin rats retained slightly more fat than did pregnant rats. The difference between pregnant and virgin rats with respect to the amount of fat in the different body parts was small in both feeding groups. Thus, the statement that fat stored at specific sites in the maternal body represents an important source of energy for use during lactation was not supported. The findings suggest that pregnancy stimulates growth of the maternal body and that chronic moderate energy restriction curtails this growth.

Estimation of total body fat and subcutaneous adipose tissue in full-term infants less than 3 months old.

Both fetal and neonatal nutrition may influence the body fat content of infants. Epidemiologic field studies would be facilitated by a simple method of measuring total body fat (TBF). The accuracy of a method, based on skinfold measurements, of estimating TBF was evaluated in 22 infants by comparing the results with those obtained by the body water dilution technique. Because the accuracy was poor, a modification was evaluated in 14 of the infants. The measurements were used to calculate the amounts of subcutaneous and nonsubcutaneous body fat. Estimates of the thickness of subcutaneous adipose tissue made with calipers and ultrasonography were compared with each other and with those obtained with Futrex 5000, a device based on the principle of near-infrared interactance. The composition of the adipose tissue in biopsy specimen from 38 infants was analyzed and contained a mean of 0.66 g of fat/cm3. Results obtained by Futrex 5000 correlated with subcutaneous adipose tissue thickness only when the latter was thin. Estimates of adipose tissue thickness by calipers were significantly higher than estimates by ultrasonography at the forearm, thigh, and calf, whereas the opposite was found at the triceps, biceps, umbilicus, and nipple. The amount of nonsubcutaneous fat, in relation to body weight minus subcutaneous fat, increased with age and was higher in girls than in boys. Skinfolds were poor predictors of TBF. However, it may be possible to predict TBF from anthropometric measurements if appropriate knowledge about the growth and development of adipose tissue in infancy are available.

Total energy expenditure of healthy Swedish women during pregnancy and lactation.

Total energy expenditure (TEE) was estimated in healthy Swedish women by the doubly labeled water method in a longitudinal study during pregnancy and lactation. Measurements were made before pregnancy (A); in gestational weeks 16-18 (B), 30 (C), and 36 (D); as well as 2 (F) and 6 (G) mo after delivery. When the results were interpreted, earlier published data regarding resting metabolic rate (RMR), energy intake, and energy in breast milk were also considered. TEE (MJ/d) and TEE/RMR were 10.5 +/- 2.2 (mean +/- SD) and 1.87 +/- 0.42 (n = 28), 9.6 +/- 2.8 and 1.65 +/- 0.67 (n = 22), 12.5 +/- 3.4 and 1.82 +/- 0.45 (n = 22), 12.2 +/- 4.1 and 1.66 +/- 0.52 (n = 19), 10.6 +/- 2.0 and 1.82 +/- 0.41 (n = 23), and 10.8 +/- 2.7 and 1.79 +/- 0.42 (n = 23) at A, B, C, D, F, and G, respectively. The results indicated that physical activity tended to be decreased during early pregnancy. Taking changes in body energy stores into consideration, it was estimated that subjects recorded 86% and 77% of their energy intakes at A and F respectively.

Energy restriction and the partitioning of energy among the costs of reproduction in rats in relation to growth of the progeny.

Compared with well-nourished controls, progeny of energy-restricted females are smaller and grow less well. The mechanisms behind these effects are frequently studied in rats but interpretation of such studies requires knowledge about the partitioning of dietary energy among the costs of reproduction and maternal needs. Energy partitioning was studied in rats fed ad libitum or 70% of ad libitum intake from 24-34 d before conception until d 14 of lactation and in virginal controls. Body composition was estimated for adult rats on d 1 of gestation, for adult rats and litters on d 1, 9 and 14 of lactation and for virginal controls after 65 d of dietary treatment. Milk production and composition were also studied. Dams in the two groups retained similar amounts of energy during pregnancy. Mobilized body energy corresponded to only 6 and 12% of the energy in milk for ad libitum-fed and restricted rats, respectively. Adult energy-restricted rats were smaller than their ad libitum-fed counterparts and thus needed less energy for maintenance. During lactation, energy-restricted rats used less energy per unit body size than ad libitum-fed rats. Pups of restricted dams were not only smaller, they also utilized milk energy less efficiently than did pups of well-fed dams.

Energy metabolism, body composition, and milk production in healthy Swedish women during lactation.

Variables of relevance to energy requirements during reproduction were studied in 23 healthy lactating Swedish women. Body composition and resting metabolic rate (RMR) were studied before pregnancy and three times postpartum. Energy intake was studied before pregnancy and, together with breast-milk production, 2 mo postpartum. The women gained 5.8 +/- 4.2 kg fat during pregnancy and their average fat content was unchanged during the first 2 mo of lactation whereas a slight loss (1.7 +/- 4.2 kg) occurred during the following 4 mo. RMR increased slightly during lactation in spite of a decrease in fat-free body weight 2 and 6 mo postpartum. Energy intake increased during lactation (280 +/- 440 kcal/d). The women produced 740 +/- 150 g breast milk/d containing 0.64 +/- 0.08 kcal/g. The results indicate that current estimates of energy needs during lactation may be too high.