Lactose drives Enterococcus expansion to promote graft-versus-host disease.

Disruption of intestinal microbial communities appears to underlie many human illnesses, but the mechanisms that promote this dysbiosis and its adverse consequences are poorly understood. In patients who received allogeneic hematopoietic cell transplantation (allo-HCT), we describe a high incidence of enterococcal expansion, which was associated with graft-versus-host disease (GVHD) and mortality. We found that Enterococcus also expands in the mouse gastrointestinal tract after allo-HCT and exacerbates disease severity in gnotobiotic models. Enterococcus growth is dependent on the disaccharide lactose, and dietary lactose depletion attenuates Enterococcus outgrowth and reduces the severity of GVHD in mice. Allo-HCT patients carrying lactose-nonabsorber genotypes showed compromised clearance of postantibiotic Enterococcus domination. We report lactose as a common nutrient that drives expansion of a commensal bacterium that exacerbates an intestinal and systemic inflammatory disease.

Rates of neutropenia in adults with influenza A or B: a retrospective analysis of hospitalised patients in South East Queensland during 2015.

Neutropenia in adult patients is often attributed to intercurrent viral infections; however, there are limited data describing the frequency or natural history of this phenomenon. We examined all patients presenting to three large hospitals in the Metro South region of South East Queensland with laboratory-confirmed influenza A or B throughout the 2015 influenza season (January-October). Four hundred and thirty-six patients were studied and 15.3% of this cohort were neutropenic (absolute neutrophil count <2.0 × 109 /L) with no identifiable cause other than the influenza. Importantly, the majority of cases were mild, with absolute neutrophil count remaining >1.0 × 109 /L. The incidence of neutropenia was significantly higher in association with influenza B than influenza A (18.3% vs 10.3%). We conclude that mild, transient neutropenia is common among patients with influenza infection and advise that it should not cause alarm or invite specific investigation unless severe or prolonged.

Blood, sweat, and tears: developing clinically relevant protein biosensors for integrated body fluid analysis.

Biosensors are being developed to provide rapid, quantitative, diagnostic information to clinicians in order to help guide patient treatment, without the need for centralised laboratory assays. The success of glucose monitoring is a key example of where technology innovation has met a clinical need at multiple levels ­ from the pathology laboratory all the way to the patient's home. However, few other biosensor devices are currently in routine use. Here we review the challenges and opportunities regarding the integration of biosensor techniques into body fluid sampling approaches, with emphasis on the point-of-care setting.

Impact of cytokine gene polymorphisms on graft-vs-host disease.

Allogeneic hematopoietic stem cell transplantation (SCT) remains the only available curative therapy for hematological malignancy. It does, however, result in significant morbidity and mortality, predominantly as a consequence of infections, leukemic relapse and graft-vs-host disease (GVHD). While differences in human leukocyte antigen (HLA) molecules between donor and host make a crucial contribution to the alloreactivity driving the donor-antihost response, the cytokine milieu consisting of molecules that both promote and regulate the alloresponse after transplantation is also critical. As such, genetic studies correlating donor and/or host cytokine polymorphisms with disease outcomes have provided useful insight into disease pathogenesis, often confirming effects that have been dissected in animal models of the disease. It is now clear that the polymorphic expression of key cytokines (particularly tumor necrosis factor and interleukin 10) has a demonstrable effect on disease outcome and overall transplant-related mortality. Consideration of the role of genetic polymorphisms in GVHD severity and procedural mortality associated with SCT will lead to improvements in patient outcome such that the addition of non-HLA genetic typing of potential donors will allow optimization of donor selection for a given recipient. This review provides a discussion of the current state of the literature regarding polymorphic expression of the key GVHD cytokines and their capacity to predict clinical disease outcome.

Glucocorticoid effects on phenylethanolamine N-methyltransferase (PNMT) in explants of embryonic rat medulla oblongata.

Although glucocorticoid hormones have important roles in the development of neurotransmitter systems in cells derived from the neural crest, it is not known whether they have parallel effects on neuronal development in the brain. To address this issue, we have established an in vitro system of fetal medulla oblongata (MO) to follow development of the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT). Embryonic MO was explanted from E13 or E18 embryos and maintained for up to 3 weeks. Successful culture of adrenergic neurons was possible only in explants taken from young embryos, since E18 explants failed to develop. In E13 explants, immunoreactivity to both PNMT and tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis, was observed. PNMT catalytic activity which was barely detectable at the time of explanation increased markedly during the first week in vitro. To study the effects of glucocorticoids on PNMT development in central neurons, MO explants were grown in glucocorticoid deficient medium in which rat serum from adrenalectomized rats was substituted for human placental serum. Addition of natural glucocorticoids, cortisol or corticosterone, or the mineralcorticoid, deoxycorticosterone, during the third culture week had no effect on PNMT activity. Dexamethasone (DEX), a synthetic glucocorticoid, also had no effect on PNMT during the first or second weeks in culture. However, addition of DEX during the third culture week resulted in a doubling of PNMT activity. However, attempts to block the DEX effect during the third week or to block the increase in PNMT activity during the first week in control cultures with the glucocorticoid receptor antagonist, dexamethasone 21-mesylate, were unsuccessful. These results suggest that PNMT in central neurons does not require glucocorticoids for ontogeny during the embryonic period. This is in contrast to PNMT in adrenal medulla which requires glucocorticoids for normal development during both the embryonic and postnatal periods. More generally, these studies suggest that development of the same neurotransmitter phenotype in brain and periphery may be differentially regulated.

Depolarizing stimuli increase tyrosine hydroxylase in the mouse locus coeruleus in culture.

The influence of membrane depolarization on the development and regulation of brain noradrenergic neurons was studied in explant cultures of the mouse locus coeruleus (l.c.). Exposure to the depolarizing agents veratridine or elevated K+ significantly increased the catalytic activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. The effects of veratridine were prevented by tetrodotoxin, suggesting that transmembrane Na+ influx was necessary for the rise in TH. Morphometric analysis indicated that the rise in TH activity was not accompanied by altered TH-positive cell number or cell diameter. Rather, TH fluorescence intensity increased in each neuron, suggesting that depolarization increased TH per neuron. Immunoblot and densitometric analysis indicated that depolarization did, indeed, increase TH immunoreactive protein. Moreover, depolarization elevated enzyme activity in cultured neurons expressing the normal developmental increase in TH, as well as those in which plateau levels had already been attained. We conclude that depolarization and/or Na+ influx regulates a critical transmitter macromolecule in brain neurons, as in the periphery, by altering enzyme molecule number.

Selective expression of high-affinity uptake of catecholamines by transiently catecholaminergic cells of the rat embryo: studies in vivo and in vitro.

Transient expression of catecholaminergic phenotypic traits is a widespread phenomenon during embryonic development in mammals, occurring in cells of the embryonic gut mesenchyme, in ventrolateral portions of the neural tube, cells of cranial sensory and dorsal root ganglia, and in the embryonic pancreas. In the current study the manifestation of the catecholamine (CA) phenotype in these populations has been further defined. Specifically, the existence of the high-affinity uptake process for CAs in these populations has been investigated. By combining the techniques of radioautography following accumulation of [3H]norepinephrine (3H-NE) and [3H]dopamine (3H-DA) with immunohistochemical detection of tyrosine hydroxylase (T-OH), it has been possible to demonstrate simultaneously CA accumulation by T-OH-positive gut cells. Uptake of 3H-NE was first detected in T-OH-positive cells of the gut on gestational day 12.5 (E12.5). By contrast, T-OH immunoreactivity was first detected on E11.5. By E13.5 virtually every T-OH-positive cell oral to the umbilical flexure was radioautographically labeled. Uptake at E13.5 displayed Michaelis-Menten saturation kinetics, had a Vmax of 35 fmole/gut/min, a Km of 1.45 microM, was blocked by desmethylimipramine (DMI), and by incubation at 4 degrees C. On subsequent gestational days, silver grains marking areas of amine concentration were found increasingly over T-OH-negative cells. A similar pattern of uptake was found in guts which had been grown in organotypic tissue culture for the purpose of eliminating extrinsic sympathetic innervation. T-OH-positive gut cells also accumulated 3H-DA. Concentration of 3H-DA was blocked by both benztropine and DMI suggesting that accumulation had properties common to both NE and DA systems. By contrast to cells of the gut, accumulation of CAs was not a property of transiently T-OH-positive cells in other locations. Therefore, specific, high-affinity uptake and retention of CAs is an additional property of transiently catecholaminergic gut cells. Appearance of CA synthetic enzymes precedes the appearance of the CA storage process in cells of the gut. Persistence of the uptake process after the loss of detectable T-OH suggests continued viability of the population. The absence of CA accumulation by other T-OH-positive cells suggests basic molecular differences among the various populations.

Influence of ACTH on tyrosine hydroxylase activity in the locus coeruleus of mouse brain.

Tyrosine hydroxylase activity in noradrenergic neurons of the locus coeruleus was found to rise in bilaterally adrenalectomized adult mice, with maximum increase of the enzyme activity (52% above control) occurring 7 days after the surgery. No such increase of tyrosine hydroxylase activity was found in dopaminergic neurons of the substantia nigra. The increase of enzyme activity in the locus coeruleus following adrenalectomy was totally prevented by corticosterone replacement. Moreover, the adrenalectomy effect was abolished by hypophysectomy, indicating the involvement of the pituitary rather than the adrenocortical function. Chronic administration of ACTH (20 IU/kg, i.p., daily) resulted in an increase of tyrosine hydroxylase activity in the locus coeruleus, with a time course and magnitude similar to those found after adrenalectomy. Additionally, the effects of four ACTH analogs were determined. ACTH 1-24 and ACTH 4-10 were as effective as the whole ACTH molecule, whereas ACTH 4-10,7-D-Phe and ACTH 11-24 were ineffective. The effect of ACTH 4-10, a peptide fragment with no adrenocorticotrophic activity, further indicates that glucocorticoids are not involved. From these data, it appears that tyrosine hydroxylase in the locus coeruleus neurons is under the regulatory influence of pituitary ACTH. It remains to be determined whether the hormone can be transported from its pituitary origin to the locus coeruleus and exerts a direct action on the noradrenergic neurons. Regardless of the mechanism, the response of the noradrenergic neurons to pituitary activity may be an important component in physiological adaptation of the central nervous system to chronic stress.

Transient expression of selected catecholaminergic traits in cranial sensory and dorsal root ganglia of the embryonic rat.

We describe the transient expression of catecholaminergic traits in cranial sensory and dorsal root ganglia of the embryonic rat in vivo. Isolated cells expressing tyrosine hydroxylase (T-OH) immunoreactivity were initially detected in trigeminal (V) ganglion anlages as early as gestational Day 10.5 (E10.5; 18-22 somites). Neurofilament (NF) protein was also evident in V at these early stages. By E11.5 (27-30 somites) clusters of T-OH-positive cells were visible in V. Many of these cells were bipolar; others sent processes into the primitive brainstem. In addition, cells expressing T-OH were apparent in primordia of sensory ganglia serving the glossopharyngeal (IX) and vagal (X) cranial nerves. By this stage (E11.5) all cranial sensory ganglia were rich in NF protein, but immunoreactivity was confined to cellular processes rather than perikarya. By E12 (35-37 somites), only a few, faintly positive T-OH-containing cells were evident in V. However, DBH- and T-OH-positive cells were visible within the more caudal nodose and petrosal ganglia. Furthermore, isolated bipolar cells expressing T-OH were detected in rostral dorsal root ganglia at this stage. Catecholamine fluorescence could not be detected in any sensory ganglia even after maternal treatment with inhibitors of monoamine oxidase. Catecholaminergic cells were not seen at any stage in anlages of the acousticovestibular nucleus. Immunoreactive T-OH was undetectable in all ganglia by E13.5 (46-48 somites). These findings highlight the fact that transient expression of the catecholamine phenotype during development is a widespread phenomenon, evident in a variety of cell types of diverse embryonic origin.

Expression of catecholaminergic characteristics by primary sensory neurons in the normal adult rat in vivo.

Expression of catecholaminergic characteristics by primary sensory neurons was examined in the vagal nodose and glossopharyngeal petrosal ganglia of the normal adult rat in vivo. Catecholaminergic phenotypic expression was documented by immunocytochemical localization of tyrosine hydroxylase (TyrOHase; EC 1.14.16.2), radiochemical assay of specific TyrOHase catalytic activity, and cytochemical localization of formaldehyde-induced catecholamine fluorescence (FIF) within principal ganglion cells. The TyrOHase-containing cells exhibited morphologic features typical of primary sensory neurons, such as an initial axon glomerulus and a single, bifurcating neurite process. These cells were distinguished from TyrOHase- and FIF-positive small intensely fluorescent cells by size, morphology, and staining intensity. TyrOHase-containing neurons appeared to be insensitive to neonatal treatment with 6-hydroxydopamine, thereby distinguishing them from sympathetic neurons. Nodose and petrosal ganglia of adult rats exhibited TyrOHase catalytic activity, linear with respect to tissue concentration over a 10-fold range, indicating that the immunoreactive enzyme was functional. Transection of specific ganglionic nerve roots depleted TyrOHase catalytic activity and neuronal immunoreactivity within the petrosal ganglion, suggesting that target organ innervation regulates enzyme levels within ganglion perikarya. Our study indicates that primary sensory neurons express catecholaminergic transmitter traits in the normal adult rat. Consequently, in the periphery, catecholaminergic characters are not restricted to the sympathoadrenal axis but are expressed by functionally and embryologically diverse populations of autonomic neurons.

Development of catecholaminergic phenotypic characters in the mouse locus coeruleus in vivo and in culture.

While abundant studies have begun to elucidate ontogeny of the peripheral nervous system, molecular mechanisms underlying brain development remain obscure. To approach this problem, we initiated parallel in vivo and in vitro studies of the mouse locus coeruleus (l.c.), a brainstem noradrenergic nucleus. The catecholaminergic enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) were used to monitor phenotype expression and development. TH catalytic activity and immunocytochemical reactivity were initially detectable on gestational Day 13 (E-13) in vivo, and adult levels of activity were approximately by the third postnatal week. Immunotitration studies indicated that the developmental increase was due to accumulation of enzyme molecules and not enzyme activation. The in vivo developmental profile of DBH approximated that of TH. To begin defining regulatory mechanisms, explants of embryonic brainstem were placed in culture. Explantation on E-12, prior to expression of TH or DBH, resulted in the de novo appearance of these phenotypic characters after 4 days. Explantation on E-18, after the enzymes are already expressed, was followed by a striking sixfold rise in TH activity. Immunotitration studies revealed that the increase in TH activity in E-18 cultures was attributable to increased molecule number, reproducing the in vivo results. Moreover, the E-18 explants, cultured for 3 weeks, attained higher plateau levels of TH activity than E-12 cultures, and this differences was due to increased molecule number. Morphometric analysis indicated that 3-week E-12 cultures actually had more l.c. cells than E-18 cultures, indicating that differences in TH were not due to increased cells in the E-18 l.c. Finally, systemic study revealed that the development of TH activity in culture increased progressively from E-11 to E-12 to E-13, suggesting that critical regulatory events occur at this time. Our studies suggest that the l.c. is an excellent model for the study of brain development in vivo and in vitro. Initial phenotypic expression and dramatic development occur in culture in the absence of normal targets, normal afferent innervation and, presumably, normal humoural milieu.

Glucocorticoid influence on tyrosine hydroxylase activity in mouse locus coeruleus during postnatal development.

Administration of corticosterone (10 mg/kg, ip, twice daily for 3 days) to mice during the second week of postnatal development led to an increase of tyrosine hydroxylase (TH) activity in the locus coeruleus, but not in the substantia nigra. The corticosterone effect was observed only transiently during this developmental period. Tritiated corticosterone can bind to a cytosol fraction prepared from mouse locus coeruleus, with a specific binding capacity of 110 fmol/mg protein. There is a correlation between the ability of various steroids to increase TH activity and their binding to the cytosol glucocorticoid receptor. Cortexolone and progesterone, two antiglucocorticoids that can bind to the cytosol receptor, were found to abolish the effect of corticosterone in increasing TH activity. It appears that the noradrenergic neurons in the locus coeruleus may be target cells for glucocorticoids, and that the glucocorticoid effect on TH may be a receptor-mediated mechanism.

Influence of adrenal epinephrine on postnatal development of tyrosine hydroxylase activity in the superior cervical ganglion.

The effects of bilateral adrenalectomy on postnatal development of tyrosine hydroxylase (TH) activity in the superior cervical ganglion were examined in the rat. Adrenalectomy on day 12 blocked further developmental rise of TH activity, while adrenalectomy on day 7 did not affect normal development of enzyme activity when examined on day 12. The effect of adrenalectomy could be reversed by replacement with daily injections of epinephrine, but not by similar replacement with corticosterone. Chronic administration of SKF 64139, a specific inhibitor of adrenal phenylethanolamine N-methyl-transferase activity that depletes circulating epinephrine levels, also led to a cessation of normal development of TH activity. Similar to adrenalectomy, the effect of SKF 64139 could be observed only after 2 weeks of postnatal development. From these data, it appears that after 2 postnatal weeks, further developmental rise of TH activity in the sympathetic ganglion is an epinephrine-dependent process.

Adrenal influence on tyrosine hydroxylase activity in superior cervical ganglion.

The involvement of adrenal hormones as regulatory factors in maintaining physiological levels of tyrosine hydroxylase (TH) was examined in mouse superior cervical ganglion. Following bilateral adrenalectomy, TH activity in the ganglion fell at a slow but steady rate, reaching 60-65% of the control levels after 2 weeks. Decentralization is known also to reduce TH activity in the ganglion. The effects of adrenalectomy and decentralization were therefore compared, and they were found to be additive, indicating different mechanisms in the two cases. The reduction of TH activity following adrenalectomy was not prevented by replacement with corticosterone (0.5 mg/kg, daily). However, replacement with epinephrine (4 mg/kg, daily) completely prevented the fall of TH activity in adrenalectomized animals. Isoproterenol, a beta-adrenergic receptor agonist, was as effective as epinephrine in preventing the reduction of TH activity following adrenalectomy. Furthermore, in intact animals, chronic administration of SKF 64139, an inhibitor of adrenal PNMT which depletes circulating epinephrine levels, also reduced ganglionic TH activity to the same level as that after adrenalectomy. These results indicate that epinephrine, but not corticosterone, is the adrenal factor required for physiological maintenance of normal levels of TH in the superior cervical ganglion.