Effects of dietary rumen-protected choline supplementation to periparturient dairy cattle on inflammation and metabolism in mammary and liver tissue during an intramammary lipopolysaccharide challenge.

The objective of this experiment was to examine the effects of supplementation and dose of rumen-protected choline (RPC) on markers of inflammation and metabolism in liver and mammary tissue during an intramammary lipopolysaccharide (LPS) challenge. Parous Holstein cows were blocked by calving month and randomly assigned within block to receive 45 g/d of RPC (20.4 g/d of choline ions; CHOL45), 30 g/d of RPC (13.6 g/d of choline ions; CHOL30), or no RPC (CON) as a top-dress starting 24 d before expected calving until 21 d postpartum. Cows were alternately assigned within treatment group to either receive an intramammary LPS challenge (200 µg in each rear quarter; Escherichia coli O111:B4) or not at 17 DIM (CHOL45, n = 9; CHOL45-LPS, n = 9; CHOL30, n = 11; CHOL30-LPS, n = 10; CON, n = 10; CON-LPS, n = 9). Hepatic and mammary tissues were collected from all cows on d 17 postpartum. Hepatic and mammary tissues were collected at ∼7.5 and 8 h, respectively, after the LPS challenge. An additional mammary biopsy was conducted on LPS-challenged cows (CHOL45-LPS, CHOL30-LPS, and CON-LPS) at 48 h postchallenge. Hepatic and mammary RNA copy numbers were quantified for genes involved in apoptosis, methylation, inflammation, oxidative stress, and mitochondrial function using NanoString technology. Targeted metabolomics was conducted only on mammary tissue samples (both 8 and 48 h biopsies) to quantify 143 metabolites including choline metabolites, amino acids, biogenic amines and derivatives, organic acids, carnitines, and glucose. Hepatic IFNG was greater in CHOL45 as compared with CON in unchallenged cows, suggesting an improvement in type 1 immune responses. Hepatic CASP3 was greater in CHOL45-LPS as compared with CON-LPS, suggesting greater apoptosis. Mammary IL6 was reduced in CHOL30-LPS cows as compared with CHOL45-LPS and CON-LPS (8 and 48 h). Mammary GPX4 and COX5A were reduced in CHOL30-LPS as compared with CON-LPS (8 h), and SDHA was reduced in CHOL30-LPS as compared with CON-LPS (8 and 48 h). Both CHOL30-LPS and CHOL45-LPS cows had lesser mammary ATP5J than CON-LPS, suggesting that dietary RPC supplementation altered mitochondrial function following LPS challenge. Treatment did not affect mammary concentrations of any metabolite in unchallenged cows, and only 4 metabolites were affected by dietary RPC supplementation in LPS-challenged cows. Mammary concentrations of isobutyric acid and 2 acyl-carnitines (C4:1 and C10:2) were reduced in CHOL45-LPS as compared with CHOL30-LPS and CON-LPS. Taken together, reductions in medium- and short-chain carnitines along with an increase in long-chain carnitines in mammary tissue from CHOL45-LPS cows suggests less fatty acid entry into the ß oxidation pathway. Although the intramammary LPS challenge profoundly affected markers for inflammation and metabolism in liver and mammary tissue, dietary RPC supplementation had minimal effects on inflammatory markers and the mammary metabolome.

Effects of dietary rumen-protected choline supplementation to periparturient dairy cattle on inflammation, metabolism, and performance during an intramammary lipopolysaccharide challenge.

Recent studies have suggested that dietary rumen-protected choline (RPC) supplementation can modulate immune function, attenuate inflammation, and improve performance in periparturient dairy cattle; however, this has yet to be evaluated during a mastitis challenge. Therefore, the objective of this study was to examine the effects of supplementation and dose of RPC on metabolism, inflammation, and performance during an intramammary lipopolysaccharide (LPS) challenge. Parous Holstein cows (parity, mean ± SD, 1.9 ± 1.1 at enrollment) were blocked by calving month and randomly assigned within block to receive either 45 g/d of RPC (20.4 g/d of choline ions; CHOL45, n = 18), 30 g/d of RPC (13.6 g/d of choline ions; CHOL30, n = 21), or no RPC (CON, n = 19) as a top-dress starting 24 d before expected calving until 21 d postpartum. Cows were alternately assigned within treatment group to either receive an intramammary LPS challenge (200 µg in each rear quarter; Escherichia coli O111:B4) or not at 17 DIM. Before the challenge, CHOL45 and CHOL30 cows produced 3.4 and 3.8 (±1.2 SED) kg/d more milk than CON, respectively. Dietary RPC supplementation did not mitigate the milk loss associated with the intramammary LPS challenge; however, CHOL45 and CHOL30 cows produced 3.1 and 3.5 (±1.4 SED) kg/d more milk than CON, respectively in the carryover period (22 to 84 DIM). Dietary RPC supplementation enhanced plasma ß-hydroxybutyrate (BHB) concentrations before the LPS challenge, and increased plasma nonesterified fatty acids (NEFA) and acetylcarnitine concentrations during the LPS challenge, potentially reflecting greater adipose tissue mobilization, fatty acid transport and oxidation. Aside from trimethylamine N-oxide and sarcosine, which were increased in CHOL45-LPS as compared with CON-LPS, most other choline metabolite concentrations in plasma were unaffected by treatment, likely because more choline was being secreted in milk. Plasma lactic acid concentrations were decreased in CHOL45-LPS and CHOL30-LPS as compared with CON-LPS, suggesting a reduction in glycolysis or an enhancement in the flux through the lactic acid cycle to support gluconeogenesis. Plasma concentrations of fumaric acid, a byproduct of AA catabolism and the urea cycle, were increased in both choline groups as compared with CON-LPS during the LPS challenge. Cows in the CHOL45 group had greater plasma antioxidant potential before the LPS challenge and reduced plasma methionine sulfoxide concentrations during the LPS challenge compared with CON-LPS, suggesting an improvement in oxidant status. Nevertheless, concentrations of inflammatory markers such as haptoglobin and tumor necrosis factor α (TNFα) were not affected by treatment. Taken together, our data suggest that the effects of dietary RPC supplementation on milk yield could be mediated through metabolic pathways and are unlikely to be related to the resolution of inflammation in periparturient dairy cattle. Lastly, dose responses to dietary RPC supplementation were not found for various economically important outcomes including milk yield, limiting the justification for feeding a greater dietary RPC dose in industry.

The effect of J5 bacterins on clinical, behavioral, and antibody response following an Escherichia coli intramammary challenge in dairy cows at peak lactation.

Vaccination against coliform mastitis has become part of mastitis control programs in the past 3 decades, as a means of reducing the severity of clinical mastitis. Our study objective was to evaluate the effect of 2 commercially available vaccines on clinical, behavioral, and antibody response following Escherichia coli intramammary challenge in cows near peak lactation. Cows (n = 12 per group) were vaccinated with vaccine 1 (V1) or vaccine 2 (V2) at dry-off, 21 d pre-calving, and 14 d post-calving. Twelve cows served as unvaccinated controls (CTL). Cows were challenged with E. coli in a rear quarter at approximately 100 d in milk. Milk samples were collected pre- and post-challenge to enumerate E. coli and determine somatic cell count. Serum was collected before each vaccination and at d 0, 1, 2, 3, 6, 30, and 60 relative to challenge, to study antibody response. Milk IgA and tumor necrosis factor-α concentrations were determined in whey. Vaginal temperature, cow activity, and milk yield and components were monitored post-challenge. Bacterial count, somatic cell score, milk yield and component decline, vaginal temperature, activity measures, and antibody and cytokine response were analyzed for treatment differences. The effects of parity, breed, and a repeated measure of time were also tested. Seven cows had to be removed from the study post-challenge for antibiotic treatment (CTL and V1, n = 3 each; V2, n = 1), 2 of which were euthanized (both CTL). Vaccinated cows exhibited fever (vaginal temperature ≥39.4°C) 3 h earlier than CTL cows, but we found no differences between treatments for bacterial count, somatic cell score, or milk yield reduction. Vaccinated cows spent more time lying per rest bout 2 d post-challenge, but total daily lying time was not different from CTL cows during the 7 d post-challenge. The vaccines differed in antibody response: V1 cows had greater serum IgG1 and IgG2 post-challenge. A parity effect was also evident: primiparous cows had lower bacterial counts, somatic cell score and a smaller milk yield decline than multiparous cows, but also had lower antibody production. Immunization with either J5 bacterin did not reduce clinical signs of mastitis in cows challenged at 100 d in milk, demonstrating that the effects of J5 vaccination had diminished at peak lactation.

Differential feeding behavior and neuronal responses to CCK in obesity-prone and -resistant rats.

Deficits in satiation signals are strongly suspected of accompanying obesity and contributing to its pathogenesis in both humans and rats. One such satiation signal is cholecystokinin (CCK), whose effects on food intake are diminished in animals adapted to a high fat diet. In this study, we tested the hypothesis that diet-induced obese prone (OP) rats exhibit altered feeding and vagal responses to systemic (IP) administration of CCK-8 compared to diet-induced obese resistant (OR) rats. We found that CCK (4.0 microg/kg) suppressed food intake significantly more in OP than OR rats. To determine whether enhanced suppression of feeding is accompanied by altered vagal sensory responsiveness, we examined dorsal hindbrain expression of Fos-like immunoreactivity (Fos-Li) following IP CCK injection in OP and OR rats. After 4.0 microg/kg CCK, there were significantly more Fos-positive nuclei in the NTS of OP compared to OR rats. Treatment with 8.0 microg/kg CCK resulted in no significant difference in food intake or in Fos-Li between OP and OR rats. Also, we found that OP rats were hyperphagic on a regular chow diet and gained more weight compared to OR rats. Finally OP rats had decreased relative fat pad mass compared to OR rats. Collectively, these results show that OP rats exhibit a different behavioral and vagal neuronal responses to CCK than OR rats.

NMDA NR2 receptors participate in CCK-induced reduction of food intake and hindbrain neuronal activation.

Previous work has shown that blockade of NMDAR by non-competitive (MK-801) and competitive (AP5) antagonists increase food intake by acting in the dorsal hindbrain. NMDAR are heteromeric complexes composed of NR1, NR2 and NR3 subunits. Competitive NR2B antagonists potently increase feeding when injected into the hindbrain. NR2 immunoreactivity is present in the hindbrain, vagal afferents and enteric neurons. NMDA receptors expressed on peripheral vagal afferent processes in the GI tract modulate responsiveness to GI stimuli. Therefore, it is possible that peripheral as well as central vagal NMDA receptors participate in control of food intake. To examine this possibility, we recorded intake of rodent chow, a palatable liquid food (15% sucrose), and non-nutrient (0.2% saccharin) solutions following intraperitoneal (i.p.) administration of D-CPPene, a competitive NMDA receptor antagonist that is selective for binding to the NR2B/A channel subunit. To assess participation of peripheral NMDA receptors in postoral satiation signals, we examined the ability of D-CPPene to attenuate reduction of feeding and hindbrain Fos expression following IP CCK administration. IP D-CPPene (2, 3 mg/kg) produced a significant increase in sucrose and chow intake but not saccharin. Pretreatment with D-CPPene (2 mg/kg) reversed CCK (2 microg/kg)-induced inhibition of sucrose intake, and attenuated CCK-induced Fos-Li in the dorsal hindbrain. These results confirm that antagonism of hindbrain NMDA receptors increases food intake. In addition our results suggest that NMDA receptors outside the hindbrain, perhaps in the periphery, participate in vagally mediated, CCK-induced reduction of food intake and NTS neuron activation.

Simulation of keratoconus observation in photorefraction.

In the recent years, keratoconus (KC) has increasingly gained attention due to its treatment options and to the popularity of keratorefractive surgery. This paper investigates the potential of identification of KC using photorefraction (PR), an optical technique that is similar to objective retinoscopy and is commonly used for large-scale ocular screening. Using personalized eye models of both KC and pre-LASIK patients, computer simulations were performed to achieve visualization of this ophthalmic measurement. The simulations are validated by comparing results to two sets of experimental measurements. These PR images show distinguishable differences between KC eyes and eyes that are either normal or ametropic. The simulation technique with personalized modeling can be extended to other ophthalmic instrument developments. It makes possible investigation with the least number of real human subjects. The application is also of great interest in medical training.

The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin.

The plant blue light receptor, phot1, a member of the phototropin family, is a plasma membrane-associated flavoprotein that contains two ( approximately 110 amino acids) flavin-binding domains, LOV1 and LOV2, within its N terminus and a typical serine-threonine protein kinase domain at its C terminus. The LOV (light, oxygen, and voltage) domains belong to the PAS domain superfamily of sensor proteins. In response to blue light, phototropins undergo autophosphorylation. E. coli-expressed LOV domains bind riboflavin-5'-monophosphate, are photochemically active, and have major absorption peaks at 360 and 450 nm, with the 450 nm peak having vibronic structure at 425 and 475 nm. These spectral features correspond to the action spectrum for phototropism in higher plants. Blue light excitation of the LOV2 domain generates, in less than 30 ns, a transient approximately 660 nm-absorbing species that spectroscopically resembles a flavin triplet state. This putative triplet state subsequently decays with a 4-micros time constant into a 390 nm-absorbing metastable form. The LOV2 domain (450 nm) recovers spontaneously with half-times of approximately 50 s. It has been shown that the metastable species is likely a flavin-cysteine (Cys(39) thiol) adduct at the flavin C(4a) position. A LOV2C39A mutant generates the early photoproduct but not the adduct. Titrations of LOV2 using chromophore fluorescence as an indicator suggest that Cys(39) exists as a thiolate.

Antigenicity of passively acquired major histocompatibility antigens on T cells.

T cell blasts and lines passively acquire MHC molecules in vitro. To determine the role of these molecules in immunoregulatory reactions, we examined whether T cell lines grown on irradiated F1 spleen cells were able to supply allogeneic MHC antigens for the stimulation of T cell proliferation. Immunofluorescence analysis demonstrates that autoreactive T cell lines grown with irradiated F1 spleen cells acquire allogeneic class II molecules and subsequently lose the MHC molecules within 4 days of coculture with syngeneic cells. The proliferative response of (H-2k x H-2d)F1T cells stimulated by a T cell line grown on (H-2k x H-2d)F1 cells is inhibited by the addition of hybridoma-culture supernatants containing anti-IAd as well as anti-IEk antibodies. The proliferation of the F1 T cells to the T cell line grown on H-2k spleen cells is only affected by supernatants containing anti-IEk antibodies. To investigate the role of acquired class I MHC antigens, we examined their ability to serve as antigens for cytotoxic cells. Anti-H-2k cytotoxic T cells are generated when H-2b T cells are cultured with an H-2b-derived T cell line, only if the line has been grown on (H-2k x H-2b)F1 cells. An H-2b-derived T cell line exposed to (H-2k x H-2b)F1 cells can be lysed by anti-H-2k cytotoxic T cells from a primary MLR. Similarly, an H-2k anti-H-2b cytotoxic T cell clone will kill an H-2k-derived T cell clone grown on (H-2k x H-2b)F1 spleen cells. These results demonstrate that passively acquired class I molecules can stimulate the generation of cytotoxic T cells that lyse cells expressing the class I antigens and that passively acquired class I molecules expressed on T cells serve as the target for cytotoxic T cells.

The syngeneic T-T lymphocyte reaction (STTLR). I. Induction of primary T anti-T cell proliferative responses in T cell cultures stimulated with self- and antigen-reactive T cells.

The generally accepted "Gershonian" view of immunoregulation attributes T cell-mediated regulation of immune responses to the activities of discrete T cell subsets with specialized functions such as help, suppression, and contrasuppression. Several observations made in our laboratory are not compatible with this paradigm. For instance, careful quantitations of carrier-specific T cell help to hapten-specific B cells in an adoptive transfer system yielded complex dose-response curves that could not be explained on the basis of interactions between discrete subsets of helper and suppressor cells. Rather, the results were most easily interpreted according to a model based on the following assumptions: (1) Regulation of helper T cell activity is a dose-dependent, dynamic property of T cell populations that exhibit a high degree of connectivity (self-recognition) and (2) helper T cells have the ability to perform different functions, depending on the current activity of other interacting lymphocytes. A good example of cloned T cells capable of performing multiple immunoregulatory functions was provided by the IEk-specific self-reactive Lbd line which provided help, suppression, and contrasuppression to T cell dependent PFC responses (see Quintáns et al., 1986). Since these effects were strictly dependent on the levels of antigen-specific T cell help, we hypothesized that Lbd cells interacted with other T cells to modulate their function. In this paper, we directly test the hypothesis that activated T cells can interact directly with resting T cells and describe the proliferative component of a syngeneic T cell anti-T cell response induced by antigen and self-reactive helper and cytotoxic T cells. In a follow-up report, we will describe the effector component of the T anti-T cell response.(ABSTRACT TRUNCATED AT 400 WORDS)

Prevention of measles in Israel: implications of a long-term partial immunization program.

Following the introduction of routine measles immunization in Israel in 1967, rapid and persistent modifications in the pattern of the disease were observed, including much more limited and more widely spaced epidemics, a change in the age distribution of measles cases, and a progressively increasing herd immunity that was estimated, following the 1982 epidemic, at 91.6 percent for the first 26 generations. This pattern supports the expectation that measles can be eliminated in Israel provided a herd immunity greater than or equal to 94 percent can be achieved before the next epidemic, which is predicted for 1988-89. A logistic approach to the elimination of measles in Israel requires (a) maintenance of an immunization rate of at least 90 percent in each newborn generation; (b) identification and immunization of still susceptible children in the 1-5 year and 6-9 year age groups, to attain vaccination coverage for at least 97 percent of this population (which should result in immunity among at least 94 percent); (c) provision of similar coverage for older, susceptible individuals in selected groups of children, adolescents, and young adults at high risk; (d) disease surveillance based on an early identification of the main sources of infection and monitoring of the active foci of disease in the neighbouring territories, which are an important potential source of the introduction of infection.