Differential cytokine and chemokine expression during rejection and infection following intestinal transplantation.

BACKGROUND/OBJECTIVES: Rejection and infectious enteritis in intestinal transplant (ITx) patients present with virtually identical symptoms. Currently, the gold standard for differentiating between these two conditions is endoscopy, which is invasive and costly. Our primary aim was to identify differences in peripheral blood cytokines during episodes of acute cellular rejection (ACR) and infectious enteritis in patients with intestinal transplants. METHODS: This was a prospective, cross-sectional study involving ITx patients transplanted between 2000 and 2016. We studied 63 blood samples collected from 29 ITx patients during periods of normal (n = 24) and abnormal (n = 17) allograft function. PBMCs from whole blood samples were cultured under unstimulated or stimulated conditions with phytohemagglutinin (PHA). The supernatant from these cultures were collected to measure cytokine and chemokine levels using a 38-plex luminex panel. RESULTS: Our study found that cytokines and chemokines are differentially expressed in normal, ACR, and infectious enteritis samples under unstimulated conditions based on heatmap analysis. Although each cohort displayed distinctive signatures, only MDC (p = 0.037) was found to be significantly different between ACR and infectious enteritis. Upon stimulation of PBMCs, patients with ACR demonstrated increased immune reactivity compared to infectious enteritis; though this did not reach statistical significance. CONCLUSIONS: To our knowledge, this is the first comprehensive study comparing cytokine expression during acute rejection and infectious enteritis in intestinal transplant recipients. Our results suggest that cytokines have the potential to be used as clinical markers for risk stratification and/or diagnosis of ACR and infectious enteritis.

Behavioral dissociation of dishabituation, sensitization, and inhibition in Aplysia.

Three forms of nonassociative learning (habituation, dishabituation, and sensitization) have commonly been explained by a dual-process view in which a single decrementing process produces habituation and a single facilitatory process produces both dishabituation and sensitization. A key prediction of this view is that dishabituation and sensitization should always occur together. However, we show that dishabituation and sensitization, as well as an additional process, inhibition, can be behaviorally dissociated in Aplysia by (i) their differential time of onset, (ii) their differential sensitivity to stimulus intensity, and (iii) their differential emergence during development. A simple dual-process view cannot explain these results; rather, a multiprocess view appears necessary to account for nonassociative learning in Aplysia.

Local pH elevation mediated by the intrabacterial urease of Helicobacter pylori cocultured with gastric cells.

Helicobacter pylori resists gastric acidity by modulating the proton-gated urea channel UreI, allowing for pH(out)-dependent regulation of urea access to intrabacterial urease. We employed pH- and Ca(2+)-sensitive fluorescent dyes and confocal microscopy to determine the location, rate, and magnitude of pH changes in an H. pylori-AGS cell coculture model, comparing wild-type bacteria with nonpolar ureI-deletion strains (ureI-ve). Addition of urea at pH 5.5 to the coculture resulted first in elevation of bacterial periplasmic pH, followed by an increase of medium pH and then pH in AGS cells. No change in periplasmic pH occurred in ureI-deletion mutants, which also induced a slower increase in the pH of the medium. Pretreatment of the mutant bacteria with the detergent C(12)E(8) before adding urea resulted in rapid elevation of bacterial cytoplasmic pH and medium pH. UreI-dependent NH(3) generation by intrabacterial urease buffers the bacterial periplasm, enabling acid resistance at the low urea concentrations found in gastric juice. Perfusion of AGS cells with urea-containing medium from coculture at pH 5.5 did not elevate pH(in) or [Ca(2+)](in), unless the conditioned medium was first neutralized to elevate the NH(3)/NH(4)(+) ratio. Therefore, cellular effects of intrabacterial ammonia generation under acidic conditions are indirect and not through a type IV secretory complex. The pH(in) and [Ca(2+)](in) elevation that causes the NH(3)/NH(4)(+) ratio to increase after neutralization of infected gastric juice may contribute to the gastritis seen with H. pylori infection.

Colloidal bismuth subcitrate impedes proton entry into Helicobacter pylori and increases the efficacy of growth-dependent antibiotics.

BACKGROUND: Successful eradication of Helicobacter pylori is becoming more difficult, mainly due to emerging antibiotic resistance. Treatment regimens containing bismuth have increased efficacy, but the mechanism is unknown. Helicobacter pylori is a neutralophile adapted to survive the acidic gastric environment via acid acclimation, but demonstrates more robust growth at neutral pH. Many antibiotics used to treat H. pylori rely on bacterial growth. AIM: To investigate the mechanism of increased efficacy of bismuth-containing H. pylori treatment regimens. METHODS: RNAseq and qPCR, urease activity in permeabilised and intact bacteria, internal pH and membrane potential were measured with and without colloidal bismuth subcitrate (CBS). Bacterial survival was assessed with CBS and/or ampicillin. RESULTS: Genes involved with metabolism and growth were upregulated in the presence of CBS at acidic pH. Urease activity of permeabilised H. pylori at pH 7.4 and 4.5 decreased in the presence of CBS, but intact urease activity decreased only at acidic pH. The fall in cytoplasmic pH with external acidification was diminished by CBS. The increase in membrane potential in response to urea addition at acidic medium pH was unaffected by CBS. The impact of CBS and ampicillin on H. pylori survival was greater than either agent alone. CONCLUSIONS: Bismuth is not acting directly on urease or the urea channel. Colloidal bismuth subcitrate impedes proton entry into the bacteria, leading to a decrease in the expected fall in cytoplasmic pH. With cytoplasmic pH remaining within range for increased metabolic activity of a neutralophile, the efficacy of growth-dependent antibiotics is augmented.

The binding selectivity of vonoprazan (TAK-438) to the gastric H+, K+ -ATPase.

BACKGROUND: The gastric H(+) ,K(+) -ATPase is the preferred target for acid suppression. Until recently, the only drugs that effectively inhibited this ATPase were the proton pump inhibitors (PPIs). PPIs are acid-activated prodrugs that require acid protection. Once acid-activated, PPIs bind to cysteines of the ATPase, resulting in covalent, long-lasting inhibition. The short plasma half-life of PPIs and continual de novo synthesis of the H(+) ,K(+) -ATPase result in difficulty controlling night-time acid secretion. A new alternative to PPIs is the pyrrolo-pyridine, vonoprazan (TAK-438), a potassium-competitive acid blocker (PCAB) that does not require acid protection. In contrast to other PCABs, vonoprazan has a long duration of action, resulting in 24-h control of acid secretion, a high pKa of 9.37 and high affinity (Ki = 3.0 ηmol/L). AIM: To determine binding selectivity of vonoprazan for the gastric H(+) ,K(+) -ATPase and to explain its slow dissociation. METHODS: Gastric gland and parietal cell binding of vonoprazan was determined radiometrically. Molecular modelling explained the slow dissociation of vonoprazan from the H(+) ,K(+) -ATPase. RESULTS: Vonoprazan binds selectively to the parietal cell, independent of acid secretion. Vonoprazan binds in a luminal vestibule between the surfaces of membrane helices 4, 5 and 6. Exit of the drug to the lumen is hindered by asp137 and asn138 in the loop between TM1 and TM2, which presents an electrostatic barrier to movement of the sulfonyl group of vonoprazan. This may explain its slow dissociation from the H(+) ,K(+) -ATPase and long-lasting inhibition. CONCLUSION: The binding model provides a template for design of novel potassium-competitive acid blockers.

Successful term pregnancy in an intestine-pancreas transplant recipient with chronic graft dysfunction and parenteral nutrition dependence: a case report.

Pregnancy after solid organ transplantation is becoming more common, with the largest recorded numbers in renal and liver transplant recipients. Intestinal transplantation is relatively new compared to other solid organs, and reports of successful pregnancy are far less frequent. All pregnancies reported to date in intestinal transplant recipients have been in women with stable graft function. The case reported here involves the first reported successful term pregnancy in an intestine-pancreas transplant recipient with chronic graft dysfunction and dependence on both transplant immunosuppression and parenteral nutrition (PN) at the time of conception. Pregnancy was unplanned and unexpected in the setting of chronic illness and menstrual irregularities, discovered incidentally on abdominal ultrasound at approximately 18 weeks' gestation. Rapamune was held, tacrolimus continued, and PN adjusted to maintain consistent weight gain. A healthy female infant was delivered vaginally at term. Medical complications during pregnancy included anemia and need for tunneled catheter replacements. Ascites and edema were improved from baseline, with recurrence of large volume ascites shortly after delivery. Successful pregnancy is possible in the setting of transplant immunosuppression, chronic intestinal graft dysfunction, and long-term PN requirement, but close monitoring is required to ensure the health of mother and child.

The effects of varying acidity on Helicobacter pylori growth and the bactericidal efficacy of ampicillin.

BACKGROUND: Penicillins inhibit cell wall synthesis; therefore, Helicobacter pylori must be dividing for this class of antibiotics to be effective in eradication therapy. Identifying growth responses to varying medium pH may allow design of more effective treatment regimens. AIM: To determine the effects of acidity on bacterial growth and the bactericidal efficacy of ampicillin. METHODS: H. pylori were incubated in dialysis chambers suspended in 1.5-L of media at various pHs with 5 mM urea, with or without ampicillin, for 4, 8 or 16 h, thus mimicking unbuffered gastric juice. Changes in gene expression, viability and survival were determined. RESULTS: At pH 3.0, but not at pH 4.5 or 7.4, there was decreased expression of ~400 genes, including many cell envelope biosynthesis, cell division and penicillin-binding protein genes. Ampicillin was bactericidal at pH 4.5 and 7.4, but not at pH 3.0. CONCLUSIONS: Ampicillin is bactericidal at pH 4.5 and 7.4, but not at pH 3.0, due to decreased expression of cell envelope and division genes with loss of cell division at pH 3.0. Therefore, at pH 3.0, the likely pH at the gastric surface, the bacteria are nondividing and persist with ampicillin treatment. A more effective inhibitor of acid secretion that maintains gastric pH near neutrality for 24 h/day should enhance the efficacy of amoxicillin, improving triple therapy and likely even allowing dual amoxicillin-based therapy for H. pylori eradication.

Cell lysis is responsible for the appearance of extracellular urease in Helicobacter pylori.

BACKGROUND: Helicobacter pylori is a neutralophilic bacterium that colonizes the acidic human gastric surface using the neutralizing capacity of a constitutively produced urease. Urease is present both in the cytoplasm and bound to the outside surface of the bacteria. The origin of the surface urease continues to be controversial. This study provides additional evidence that the origin of surface urease is cell lysis, not secretion. METHODS: H. Pylori was transformed with a plasmid encoding green fluorescent protein (GFP), a non-native cytoplasmic protein. Cultures supplemented with beta-cyclodextrin or horse serum were collected over various time periods and spun through a ficoll cushion to gently separate whole bacteria from released protein. The pellet and supernatant fractions were analyzed by fluorimetry, SDS-PAGE and Coomassie blue or Western analysis. RESULTS: GFP fluorescence and antigenic reactivity in the supernatant increased at each time point. GFP, the non-native cytoplasmic protein, and UreB, a native cytoplasmic protein, increased over time in the supernatant and both proteins were always present in the pellet fraction. UreI, an inner membrane protein, was only present in the pellet fraction. beta-galactosidase, a protein not found in H. pylori, was used as a negative control. CONCLUSIONS: Since it is unlikely that there is an intrinsic secretion system for GFP, a non-native protein, its increasing presence over time in the supernate fraction along with UreB, and retention of UreI in the pellet fraction implies that cell lysis accounts for the presence of urease on the surface of H. pylori.

Development and modulation of endogenous bursting in identified neuron R15 of juvenile Aplysia.

Evidence from a variety of both vertebrate and invertebrate preparations has demonstrated that modulation of the intrinsic firing patterns of individual neurons can have a dramatic effect on the functional output of a neural circuit. Although the mechanisms underlying the production and modulation of intrinsic firing patterns have been extensively studied in adult nervous systems, relatively little is known about how these two features of intrinsically active neurons develop. To address these issues, we have examined the development of endogenous bursting and its modulation by neuropeptides in the identified cell R15 of juvenile Aplysia. Confirming Ohmori (1981), we found that the mature parabolic bursting pattern of R15 is absent in early juvenile stages and develops only gradually over the last stage of juvenile development. We have then analyzed the modulatory effects of extracts made from the neurosecretory bag cells of Aplysia on the immature firing pattern of juvenile R15 cells. In the adult, neuroactive peptides released from the bag cells are known to intensify bursting. In juveniles, we have found that bag cell extract (BCE) can induce bursting prematurely as well as intensify immature bursts, whereas control extracts have no effect on the firing pattern of R15. These results show that the ionic currents necessary for the generation of endogenous bursting in R15 are present and can be modulated before the normal developmental expression of the burst pattern.

Developmental emergence of different forms of neuromodulation in Aplysia sensory neurons.

The capacity for neuromodulation and biophysical plasticity is a defining feature of most mature neuronal cell types. In several cases, modulation at the level of the individual neuron has been causally linked to changes in the functional output of a neuronal circuit and subsequent adaptive changes in the organism's behavioral responses. Understanding how such capacity for neuromodulation develops therefore may provide insights into the mechanisms both of neuronal development and learning and memory. We have examined the development of multiple forms of neuromodulation triggered by a common neurotransmitter, serotonin, in the pleural sensory neurons of Aplysia californica. We have found that multiple signaling cascades within a single neuron develop sequentially, with some being expressed only very late in development. In addition, our data suggest a model in which, within a single neuromodulatory pathway, the elements of the signaling cascade are developmentally expressed in a "retrograde" manner with the ionic channel that is modulated appearing early in development, functional elements in the second messenger cascade appearing later, and finally, coupling of the second messenger cascade to the serotonin receptor appearing quite late. These studies provide the characterization of the development of neuromodulation at the level of an identified cell type and offer insights into the potential roles of neuromodulatory processes in development and adult plasticity.

Immunization schedules for influenza.

Although on an individual basis and for some selected closed groups immunization against influenza has proved its value over the last 30 years, it has not resulted in prevention of epidemics. The impact of vaccination on national morbidity and mortality statistics has been disappointing. The reasons for this apparent failure of disease control by immunization are discussed. It is concluded that influenza vaccination has not been practised on a large enough scale to achieve an obvious effect on the spread of the viruses in open communities. The groups of people who deserve special attention in order to minimize the damages caused by influenza epidemics are considered. Advantages and disadvantages of available types of vaccine, methods and routes of administration are assessed. It is suggested that systematic application of present knowledge would probably have more impact in the near future than efforts to increase vaccine efficacy. The main current deficiencies are identified as the difficulty of producing adequate supplies of properly constituted vaccines at short notice and the general reluctance to accept annual large-scale immunization programmes. In the view of the authors these logistic and administrative problems could be resolved by long-term coordinated planning between relevant authorities and vaccine manufacturers.

A cellular analysis of inhibition in the siphon withdrawal reflex of Aplysia.

Recent behavioral experiments examining the siphon withdrawal reflex of Aplysia have revealed inhibitory effects of strong tail shock, a stimulus commonly used as an unconditioned stimulus in studies of associative and nonassociative learning in Aplysia. We utilized a reduced preparation to perform a cellular analysis of tail shock-induced inhibition in the siphon withdrawal reflex. First, we carried out behavioral studies that showed that the reduced preparation exhibits a siphon withdrawal reflex to water jet stimuli, and that tail shock produces inhibitory behavioral effects comparable to those in the intact animal: (1) strong shock produces transient inhibition of nonhabituated responses, and (2) a habituated response is facilitated by weak shock, but not by strong shock, suggesting that increasing tail shock intensity recruits the inhibitory process that competes with facilitation of habituated reflexes. Next, we carried out cellular studies that showed that the amplitude of the complex EPSP in siphon motor neurons elicited by water jet stimuli to the siphon also exhibits the inhibitory patterns produced by tail shock: (1) the nondecremented complex EPSP (a neural correlate of a nonhabituated siphon withdrawal reflex) is significantly inhibited 90 sec after strong tail shock and recovers to preshock levels 10 min later, and (2) the decremented complex EPSP (a neural correlate of a habituated reflex) is significantly facilitated by weak shock, but is not facilitated by strong shock. In addition to the complex EPSP, we simultaneously examined the monosynaptic connection between siphon sensory neurons and siphon motor neurons. The monosynaptic EPSP does not show the pattern of inhibitory modulation by tail shock exhibited by the siphon withdrawal reflex and the complex EPSP: (1) the nondecremented monosynaptic EPSP is not inhibited 90 sec after strong shock, but tends to be above preshock levels; and (2) the decremented monosynaptic EPSP is facilitated by weak as well as strong tail shock. Our results suggest that an important component of the inhibitory process triggered by strong tail shock is mediated by neural elements presynaptic to the siphon motor neurons. Because modulation of the monosynaptic connection between identified siphon sensory and siphon motor neurons does not parallel the tail shock-induced inhibitory patterns observed in the siphon withdrawal reflex and in the complex EPSP, other synaptic connections are likely to play an important role in mediating tail shock-induced inhibition in the siphon withdrawal reflex.

Development of learning and memory in Aplysia. III. Central neuronal correlates.

The defensive withdrawal reflex of the mantle organs of Aplysia californica has 2 major components, siphon withdrawal and gill withdrawal. In the previous paper of this series (Rankin and Carew, 1987), the development of 2 forms of nonassociative learning, habituation and dishabituation, was examined in the siphon withdrawal component of the reflex. In the present study we examined these same forms of learning in the gill withdrawal component of the reflex. The purpose of these experiments was 2-fold: to examine the development of learning in the other major component of the reflex; and to establish preparations in which it is possible to carry out a cellular analysis of the development of learning in the CNS. We first established that the gill withdrawal reflex in intact animals exhibited significant habituation in response to repeated tactile stimulation of the siphon and significant dishabituation in response to tail shock. We next determined the contribution of the CNS to the gill withdrawal reflex by surgically removing the abdominal ganglion from intact animals. Using the same stimulus intensity (4 mg) that produced habituation in the previous experiments, we found that the CNS accounted for approximately 95% of the reflex. Finally, we developed 2 preparations that allowed us to relate behavioral observations of learning directly to neural plasticity exhibited in the CNS. In a semi-intact preparation gill withdrawal was behaviorally measured as in the intact animal, but tactile stimulation of the siphon (to produce habituation) and shock to the tail (to produce dishabituation) were replaced by electrical stimulation of the siphon nerve and left connective, respectively. Stimulation parameters were matched to produce behavioral responses comparable with those in the intact animal. In an isolated CNS preparation the same nerve stimuli were used as in the semi-intact preparation, but the response measure used was the evoked neural discharge recorded in an efferent nerve innervating the gill. Both preparations exhibited response decrement and facilitation that was quantitatively as well as qualitatively similar to that observed in intact animals, indicating that 2 simple forms of learning exhibited by the gill withdrawal reflex in juvenile Aplysia can be localized to neural circuits within the abdominal ganglion.

The role of GSK3beta in regulating neuronal differentiation in Xenopus laevis.

The serine threonine protein kinase encoded by the shaggy locus has been implicated in neurogenesis in Drosophila. In vertebrates, the shaggy homolog, GSK3beta, is involved in early pattern formation, specifically in setting up the dorsal ventral axis. In the present study we have cloned the Xenopus homolog of the shaggy kinase and show (1) that GSK3beta is expressed in the right time and place to play a role in primary neurogenesis in Xenopus; (2) that overexpression of wild-type GSK3beta leads to a decrease in the number of primary neurons; (3) that inhibition of endogenous GSK3beta activity with overexpression of a dominant negative GSK3beta construct leads to an increase in the number of primary neurons; and (4) that GSK3beta inhibits the ability of neurogenin and NeuroD to produce ectopic tubulin expression, but does not inhibit the ability of neurogenin to produce ectopic NeuroD. On the basis of these data we propose that GSK3beta inhibits the function of NeuroD and therefore prevents neuronal differentiation at a relatively late stage in the developmental pathway.

Development of behavior and learning in Aplysia.

A set of fundamental issues in neuroethology concerns the neural mechanisms underlying behavior and behavioral plasticity. We have recently analyzed these issues by combining a simple systems approach in the marine mollusc Aplysia with a developmental analysis aimed at examining the emergence and maturation of different forms of behavior and learning. We have focussed on two kinds of questions: 1) How are specific neural circuits developmentally assembled to mediate different types of behaviors? and 2) how is plasticity integrated with these circuits to give rise to different forms of learning? From our analysis of the development of learning and memory in Aplysia, several themes have emerged: 1) Different forms of learning emerge according to different developmental timetables. 2) Cellular analogs of learning have the same developmental timetables as their respective forms of behavioral learning. 3) An analysis of non-decremented responses prior to the emergence of sensitization reveals a novel inhibitory process on both behavioral and cellular levels. 4) Sensitization emerges simultaneously in diverse response systems, suggesting an underlying general process. 5) A widespread proliferation of central neurons occurs in the same developmental stage as the emergence of sensitization, raising the possibility that some aspect of the trigger for neuronal proliferation may also contribute to the expression of sensitization.

Multiple forms of non-associative plasticity in Aplysia: a behavioural, cellular and pharmacological analysis.

A complete understanding of the cellular mechanisms underlying the formation of associations between stimuli, as occurs during classical conditioning, requires an understanding of the non-associative effects of the individual stimuli. The siphon withdrawal reflex of Aplysia exhibits both non-associative and associative learning when a tactile stimulus to the siphon serves as a conditioned stimulus, and tail shock serves as an unconditioned stimulus. In this chapter we describe experiments which examine the non-associative effects of tail shock at three different levels of analysis. At a behavioural level we found that the magnitude, and even the sign of reflex modulation induced by tail shock depended critically on three parameters: (i) the state of the reflex (habituated or non-habituated); (ii) the strength of the tail shock, and (iii) the time of testing after tail shock. Specifically, when non-habituated responses produced by water jet stimuli to the siphon were examined, tail shock produced transient inhibition 90 s later; facilitation of non-habituated responses (sensitization) only emerged after a considerable delay of 20-30 min. When habituated responses were examined, tail shock produced immediate facilitation (dishabituation); the amount of facilitation was inversely related to the strength of tail shock, with stronger shock producing no dishabituation. At a cellular level it was found that the complex excitatory postsynaptic potential (EPSP) in siphon motor neurons produced by water jet stimuli to the siphon provides a reliable cellular correlate of several of the non-associative effects of tail shock that we observe behaviourally. When non-decremented complex EPSPS were examined, strong tail shock produced transient inhibition at a test 90 s after shock.(ABSTRACT TRUNCATED AT 250 WORDS)