Acute phase reaction to lipopolysaccharide-induced mastitis in early lactation dairy cows fed nitrogenic, glucogenic, or lipogenic diets.

The availability of certain macronutrients is likely to influence the capacity of the immune system. Therefore, we investigated the acute phase response to intramammary (i.mam.) lipopolysaccharide (LPS) in dairy cows fed a nitrogenic diet (n = 10) high in crude protein, a glucogenic diet (n = 11) high in carbohydrates and glucogenic precursors, or a lipogenic diet (n = 11) high in lipids. Thirty-two dairy cows were fed one of the dietary concentrates directly after calving until the end of trial at 27 ± 3 days in milk (mean ± standard deviation). In wk 3 of lactation, 20 µg of LPS was i.mam. injected in one quarter, and sterile NaCl (0.9%) in the contralateral quarter. Milk samples of the LPS-challenged and control quarter were taken hourly from before (0 h) until 9 h after LPS challenge and analyzed for milk amyloid A (MAA), haptoglobin (HP), and IL-8. In addition, blood samples were taken in the morning, and composite milk samples at morning and evening milkings, from 1 d before until 3 d after LPS challenge, and again on d 9, to determine serum amyloid A (SAA) and HP in blood, and MAA and HP in milk. The mRNA abundance of various immunological and metabolic factors in blood leukocytes was quantified by quantitative reverse-transcription PCR from samples taken at -18, -1, 6, 9, and 23 h relative to LPS application. The dietary concentrates did not affect any of the parameters in blood, milk, and leukocytes. The IL-8 was increased from 2 h, HP from 2 to 3 h, and MAA from 6 h relative to the LPS administration in the milk of the challenged quarter and remained elevated until 9 h. The MAA and HP were also increased at 9 h after LPS challenge in whole-udder composite milk, whereas HP and SAA in blood were increased only after 23 h. All 4 parameters were decreased again on d 9. Similar for all groups, the mRNA abundance of HP and the heat shock protein family A increased after the LPS challenge, whereas the mRNA expression of the tumor necrosis factor α and the leukocyte integrin ß 2 subunit (CD18) were decreased at 6 h after LPS challenge. The glucose transporter (GLUT)1 mRNA abundance decreased after LPS, whereas that of the GLUT3 increased, and that of the GLUT4 was not detectable. The mRNA abundance of GAPDH was increased at 9 h after LPS and remained elevated. The acute phase protein response was detected earlier in milk compared with blood indicating mammary production. However, immunological responses to LPS were not affected by the availability of specific macronutrients provided by the different diets.

Effect of different dietary regimens at dry-off on performance, metabolism, and immune system in dairy cows.

Concentrate withdrawal and feed restriction are commonly used to reduce milk production and to facilitate dry-off, but may impair immune function in dairy cows. We investigated the effect of feed rations providing different amounts of nutrients in combination with feed restriction on performance, endocrine, and metabolic responses, as well as on leukocyte function before and after abrupt dry-off. Forty-three cows were studied from d 12 before until d 6 after dry-off (56 d before scheduled calving). Cows were fed experimental concentrates rich in crude protein (nitrogenic, n = 14), glucogenic precursors (glucogenic, n = 14), or lipids (lipogenic, n = 15). On d 3 before dry-off, total feed allowance was restricted to 50% in half of the animals of each dietary group, whereas feed allowance remained unchanged in the other animals. Performance parameters (milk yield, milk composition, and dry matter intake) were recorded, and daily blood and milk samples were taken and analyzed for various metabolic and endocrine parameters. Additionally, activity and mRNA abundance of several genes in leukocytes were measured at selected time points before and after feed restriction and dry-off, respectively. Feed restriction immediately resulted in a negative energy balance and decreased milk production. Concomitantly, concentrations of nonesterified fatty acids increased, whereas insulin, insulin-like growth factor-1, and glucagon decreased. After dry-off, energy balance turned positive and plasma nonesterified fatty acids decreased. Plasma glucose, insulin, and insulin-like growth factor-1 concentrations increased in all groups after dry-off. Glucose, insulin, and glucagon concentrations in plasma were higher in nonrestricted compared with restricted animals after dry-off. The experimental concentrate types marginally affected the investigated metabolic and endocrine factors, with the exception of elevated milk and plasma urea concentrations in cows fed the nitrogenic concentrate. Chemotactic and phagocytic activity of leukocytes were not affected by diets, feed restriction, or dry-off. Likewise, blood leukocyte mRNA abundance encoding for tumor necrosis factor α (TNF), heat shock protein family A (HSP70), and the glucose transporters (GLUT) 1 and 3 remained unchanged throughout the study period. Overall, the short-term negative energy balance induced by feed restriction was temporarily accompanied by metabolic adaptations, but did not alter the studied factors related to the immune system. Metabolic and endocrine adaptations supporting milk synthesis were continued during the first days after dry-off despite cessation of milking. Thus, the abrupt dry-off resulted in a short-term increase of glucose and triglyceride concentrations, with a delayed endocrine response to re-establish nutrient homeostasis in blood.

Chiropractic care and ochronotic arthropathy.

OBJECTIVE: To discuss the case of a patient with ochronotic arthropathy whose symptoms were treated with chiropractic care. An emphasis is placed on this condition's radiographic features. CLINICAL FEATURES: A 59-year-old woman with pain in her low back, right knee, and left ankle sought chiropractic evaluation. Black pigmentation was found in the sclera of both eyes, and homogentisic acid was present in the urine. Orthopedic evaluation revealed uncomplicated, nonspecific joint pain, and radiographs demonstrated characteristic spinal changes. INTERVENTION AND OUTCOME: The patient was treated with chiropractic manipulation, physiotherapy modalities, bracing, and exercises. This type of therapy was successful in reducing the symptoms and helped decrease the severity and frequency of acute exacerbations. CONCLUSION: Ochronotic arthropathY is a rare metabolic disorder that can be diagnosed from spinal radiographs. Chiropractic care is an appropriate tool for reducing its symptomatology.

The keratinocyte-derived cytokine IL-7 increases adhesion of the epidermal T cell subset to the skin basement membrane protein laminin-5.

Human epidermis contains a subset of epidermal T cells that can mount an immune response by migrating through the skin and into the peripheral lymphnodes to proliferate before re-entering the epidermis. The cytokine IL-7 is shown to be localized to the basement membrane of normal human skin. Furthermore, culturing in the presence of IL-7 causes increased adhesion of epidermal T cells but not peripheral blood T cells to the major epidermal basement membrane protein, laminin-5. The mechanism for increased T cell adhesion to laminin-5 is due, at least in part, to an increase in the cell surface expression of the integrin alpha3beta1. Epidermal T cells cultured in IL-7 that are strongly adherent to laminin-5 are shown by flow cytometry to consist of a variety of subsets; therefore, the increase in cell adhesion is not due to an outgrowth of one T cell subset during culturing. We hypothesize that in vivo, exposure to IL-7 is required for epidermal T cell adhesion to laminin-5.

Intraocular pressure measurement in the conscious rat.

PURPOSE: To develop a method for measurement of intraocular pressure in conscious, unsedated rats. METHODS. The animal was gently held with a thick fabric mitten, topical anesthetic drops were instilled and the Tono-Pen was applied to the cornea. RESULT: Measurements in a total of 51 animals did not differ significantly among four strains studied: the overall mean intraocular pressure+/-standard deviation was 13.0+/-1.2 mm Hg. Several intraocular pressure tolerance limits were calculated from this conscious rat data to provide a baseline estimate for future studies. CONCLUSIONS: This measurement method in conscious rats may contribute to making this widely used laboratory animal available for intraocular pressure research.

Preoperative predictors of cost in Medicare-age patients undergoing coronary artery bypass grafting.

BACKGROUND: Identification of preoperative factors that contribute to the cost of coronary artery bypass grafting could aid in predicting the procedure's expense. In this study, 30 sociodemographic and clinical preoperative factors were examined with "survival analysis" techniques to determine characteristics related to total hospital cost. METHODS: Characteristics of all patients age 65 or older undergoing isolated coronary artery bypass grafting from July 1993 to April 1995 (n = 757) were recorded. Software was developed within the hospital's Transitions Systems, Inc, database to calculate the outcome variable of total cost. Nonparametric methods were used for the univariate analysis of the data, and the Cox proportional hazards model was used for the multivariable analysis, censoring 25 patients who died in the hospital. RESULTS: Median hospital cost from the day of the operation until discharge was $15,198. Median length of stay after the operation was 6 days. Multivariable analysis revealed that age, preoperative renal failure, history of cerebrovascular accident, low ejection fraction, and surgical urgency were independent predictors of total cost. CONCLUSIONS: This study, using an accurate representation of true hospital cost and a modeling technique that accounts for the confounding effect of in-hospital death on cost, provides a template for analysis of cost in other patient groups.

The relation of pneumothorax and other air leaks to mortality in the acute respiratory distress syndrome.

BACKGROUND: In patients with the acute respiratory distress syndrome, pneumothorax and other air leaks - any extrusion of air outside the tracheobronchial tree - have been attributed to high ventilatory pressures or volumes and linked to increased mortality. METHODS: We analyzed data from a prospective trial of aerosolized synthetic surfactant in 725 patients with the acute respiratory distress syndrome induced by sepsis. We compared the ventilatory pressures and volumes in the patients without any air leaks (the highest values during the five-day study) with the pressures and volumes in those with pneumothorax or with any air leaks (the highest values during the 16- and 24-hour periods before the complication developed). RESULTS: Fifty patients (6.9 percent) had pneumothorax and 77 (10.6 percent) had pneumothorax or other air leaks. There were no significant differences between patients with air leaks and those without air leaks in any pressure or volume examined. Overall mortality at 30 days was 40.0 percent (95 percent confidence interval, 36.4 to 43.6); among the patients with pneumothorax, it was 46.0 percent (95 percent confidence interval, 32.2 to 59.8), and among those without pneumothorax, it was 39.3 percent (95 percent confidence interval, 35.6 to 43.0; P=0.35). The mortality rate was 45.5 percent (95 percent confidence interval, 34.4 to 56.6) in the group with any air leaks and 39.0 percent (95 percent confidence interval, 35.3 to 42.8) in the group without air leaks (P=0.28). CONCLUSIONS: In patients with sepsis-induced acute respiratory distress syndrome who were receiving mechanical ventilation with conventional pressures and volumes, there were no significant correlations between high ventilatory pressures or volumes and the development of pneumothorax or other air leaks. Pneumothorax or other air leaks were not associated with a significantly increased mortality rate.

During development, 17alpha-estradiol is a potent estrogen and carcinogen.

Neonatal administration of estradiol-17beta (E2-17beta) increases the nuclear DNA content in the mouse reproductive tract. Similar responses have been demonstrated for synthetic estrogens such as diethylstilbestrol. One of the questions raised regarding environmental estrogens such as organochlorines is whether they are potent enough to result in abnormal changes such as those demonstrated by both natural and synthetic estrogens. To test this hypothesis, female BALB/c mice were treated neonatally (days 1-5) with either E2-17beta or estradiol-17alpha (E2-17alpha), an inactive stereoisomer in adult reproductive tissues. We also proposed whether neonatal administration of (E2-17alpha) was tumorigenic and whether the effects were age dependent. To answer these questions, one set each of 10 day-old treated and control mice received short-term secondary administration of E2-17beta, E2-17alpha, or cholesterol. Cervicovaginal tracts from intact BALB/c mice were examined histologically and by flow cytometry at 70 days of age and by histology alone at 18 to 22 months of age. The results include several important findings: a) like E2-17beta, neonatal E2-17alpha treatment induced persistent vaginal cornification, hypospadias, vaginal concretions, and hyperproliferation in nearly 100% of the animals regardless of the secondary treatment for most groups; b) neonatal E2-17alpha treatment increased the nuclear DNA content of cervicovaginal epithelium at one-half both the level (mean DNA index of 1.02 vs 1.04) and incidence (22 vs 46% of the animals) of E2-17beta; c) short-term secondary treatment with E2-17alpha, unlike E2-17beta, did not significantly augment the increase in DNA content (13% for E2-17alpha vs 37 and 56% for control and E2-17beta, respectively); and d) neonatal administration with E2-17alpha induced adenosquamous tumors in the reproductive tract in 25% of the animals. Therefore, the biological effects (estrogenic potency) of E2-17alpha may be age dependent.

Human, canine and murine BRCA1 genes: sequence comparison among species.

Five to ten percent of breast cancer in the western world may be attributed to the inheritance of highly penetrant mutations in the breast and ovarian cancer susceptibility gene, BRCA1. The biological function of BRCA1 and factors affecting expressivity, such as gene-environment and gene-gene interactions, may be more effectively studied in appropriate animal models. We report the cloning and sequencing of the canine and murine BRCA1 genes and contrast the sequences with human BRCA1. The amino terminal 120 residues of the gene are > 80% identical among the three species. The C-terminus is also highly conserved, containing an 80 amino acid stretch that is over 80% identical. Motifs of likely functional significance are maintained, including the amino terminal RING finger motif (amino acids 24-64) and the granin consensus sequence (1214-1223). The distribution of missense mutations and neutral polymorphisms identified in BRCA1-linked breast cancer suggests that disease associated missense mutations occur at highly conserved residues whereas polymorphisms are in regions of lower conservation. Among eighteen missense mutations with unknown consequences, seven occur in amino acids that are identical across species. Four of these seven (E1219D, A1708E, P1749R and M1775R) are also within conserved domains. Taken together, these data predict regions of the gene which may be critical for normal function.

An efficient Shine-Dalgarno sequence but not translation is necessary for lacZ mRNA stability in Escherichia coli.

The 5' ends of many bacterial transcripts are important in determining mRNA stability. A series of Shine-Dalgarno (SD) sequence changes showed that the complementarity of the SD sequence to the anti-SD sequence of 16S rRNA correlates with lacZ mRNA stability in Escherichia coli. Several initiation codon changes showed that an efficient initiation codon is not necessary to maintain lacZ mRNA stability. A stop codon in the 10th codon of lacZ increased mRNA stability. Therefore, ribosomal binding via the SD sequence but not translation of the coding region is necessary to maintain lacZ mRNA stability.

Transcriptional slippage occurs during elongation at runs of adenine or thymine in Escherichia coli.

A run of 11 adenine or thymine residues at the 5' end of an out-of-frame lacZ gene causes a high level of beta-galactosidase expression in E. coli. This effect was not observed for a run of guanine residues. Reverse transcription of mRNA isolated from E. coli containing the run of 11 A's reveals heterogeneity of transcript length while reverse transcription of mRNA isolated from S. cerevisiae containing the same gene shows no heterogeneity. Protein sequencing of the beta-galactosidase molecules derived from the out-of-frame construct containing a run of adenines reveals the addition of a lysine at the run. A new method was developed where messages small enough to allow resolution of single nucleotide differences on an acrylamide gel are electrophoresed, electroblotted onto nylon and probed. This confirmed the reverse transcription results and showed that additional residues can be added to transcripts derived from DNA containing 10 or 11 thymine residues. A mechanism for slippage is discussed where the A-U rich RNA-DNA hybrid can denature during elongation and rehybridize in an offset position, causing the addition of extra residues to the transcript.

Demonstration of IL-1 alpha, and IL-1 beta secretion by the monocytic leukemia cell line, THP-1.

This study examined the secretion of IL-1 alpha and IL-1 beta by THP-1 leukemia cells following activation with mezerein and promotion of synthesis by interferon (IFN-gamma). Interleukin-1 (IL-1) was not detected by co-mitogenic thymocyte assays of crude supernates. Isoelectrofocusing of concentrated medium showed that all biologically active IL-1 migrated at a pH of 6.8-7.2, indicating that the major secreted form was IL-1 beta. Double antibody ELISA confirmed the presence of IL-1 beta, but failed to detect IL-1 alpha in isofocused fractions. Although it appeared that THP-1 cells do not secrete IL-1 alpha; an inhibitor of thymocyte response to IL-1 was present in conditioned medium, migrated in an acidic pH range and masked the expression of biologically active rIL-1 alpha and rIL-1 beta. In contrast, IL-1 alpha was detected using a cell blotting assay. This technique permitted visualization of subpicogram levels of IL-1 when secreted by cells attached to an immunoblotting paper. Cell blotting showed that a greater proportion of attached cells incubated for 24 h in medium containing mezerein and IFN-gamma secreted IL-1 than cells in control medium. In conclusion, the amount of immunoreactive or biologically active IL-1 alpha secreted by stimulated THP-1 cells appeared to be much lower than that reported for human peripheral blood monocytes.

Studies on the mechanism of captopril induced hypotension in rats.

The mechanism of the hypotensive response to captopril was investigated in pithed and urethane anesthetized rats. Cumulative intravenous doses of captopril produced dose-dependent hypotensive responses which were correlated with blockade of the pressor response to angiotensin I. Angiotensin II responses were unaffected. Infusions of saralasin, an angiotensin receptor antagonist, inhibited the hypotensive action of captopril, the degree of antagonism being correlated with inhibition of angiotensin II pressor responses. Bilateral nephrectomy lowered blood pressure to approximately the same level as captopril and completely abolished the hypotensive effect of captopril. No evidence was obtained for the involvement of endogenous bradykinin or prostaglandins in the hypotensive action of captopril. It is concluded that in pithed and urethane anesthetized rats, captopril mediates its hypotensive response by removing the renal renin-angiotensin system, most probably via inhibition of angiotensin I - converting enzyme. The results contrast with similar studies conducted in anesthetized dogs. In this species, the mechanism of action of captopril remains for further resolution.

Rapid paper chromatographic separation of [14C] angiotensen II from some metabolites: application to organ distribution.

A rapid, inexpensive method for the separation of 5-1-isoleucyl[14C] angiotensin II (A-II) from its various metabolites has been devised. A-II was extracted from tissues with absolute methanol (recovery 96%) and paper chromatographed in a butanol-acetic acid-water (18:2:5) medium for two ascents at 60 degrees C. The resulting RF for A-II of 0.45 was then compared with the RF values of three A-II metabolites produced by enzymatic degradation of the 14C-A-II and [14C]isoleucine. Trypsin degradation produced the [14C]hexapeptide metabolite, chymotryptic degradation produced the [14C]tetrapeptide metabolite and carboxypeptidase A degradation produced the [14C]heptapeptide. Increases in temperature produced a continuous increase in RF values for all the substances examined but the resolution decreased above 60 degrees C. Similarly, increases in the temperature caused the appearance of secondary peaks with some but not all peptides. The tryptic digest (hexapeptide) and the chymotryptic digest (tetrapeptide) are apparently acid- and heat-stable under the experimental conditions. All of the peptides examined failed to produce secondary peaks when heated at neutral pH. The method was used to study the tissue distribution of 14C-A-II after intravenous injection.

Localization of 14C-angiotensin in rat brain after intravenous injection.

14C-angiotensin II (14C-AII) was infused into rats which were sacrificed 5, 15, 30, 60 min post infusion. Blood and brain samples were extracted and chromatographed on paper with standards of authentic 14C-AII and metabolites produced by the enzymatic degradation of the 14C-AII. Blood levels of both total radioactivity and 14C-angiotensin declined rapidly at first and slowly thereafter. Similarly total brain radioactivity also declined with time but 14C-angiotensin levels increased over the 60 min resulting in a large increase in the percentage of angiotensin found in the brain. Examination of the brain/blood ratio for 14C-angiotensin at each time point indicates that the brain reaches equilibrium with the blood (brain/blood = 1) at 30 min and remains unchanged at 60 min. This data suggests that intravenously injected 14C-angiotensin II can cross the blood brain barrier by a passive mechanism resulting in the binding of the hormone at central sites.