Simulation of Legionnaires' disease prospective spatiotemporal cluster detection, Allegheny County, Pennsylvania, USA.

Legionnaires' disease (LD) incidence in the USA has quadrupled since 2000. Health departments must detect LD outbreaks quickly to identify and remediate sources. We tested the performance of a system to prospectively detect simulated LD outbreaks in Allegheny County, Pennsylvania, USA. We generated three simulated LD outbreaks based on published outbreaks. After verifying no significant clusters existed in surveillance data during 2014-2016, we embedded simulated outbreak-associated cases into 2016, assigning simulated residences and report dates. We mimicked daily analyses in 2016 using the prospective space-time permutation scan statistic to detect clusters of ⩽30 and ⩽180 days using 365-day and 730-day baseline periods, respectively. We used recurrence interval (RI) thresholds of ⩾20, ⩾100 and ⩾365 days to define significant signals. We calculated sensitivity, specificity and positive and negative predictive values for daily analyses, separately for each embedded outbreak. Two large, simulated cooling tower-associated outbreaks were detected. As the RI threshold was increased, sensitivity and negative predictive value decreased, while positive predictive value and specificity increased. A small, simulated potable water-associated outbreak was not detected. Use of a RI threshold of ⩾100 days minimised time-to-detection while maximizing positive predictive value. Health departments should consider using this system to detect community-acquired LD outbreaks.

Donor age and early graft failure after lung transplantation: a cohort study.

Lungs from older adult organ donors are often unused because of concerns for increased mortality. We examined associations between donor age and transplant outcomes among 8860 adult lung transplant recipients using Organ Procurement and Transplantation Network and Lung Transplant Outcomes Group data. We used stratified Cox proportional hazard models and generalized linear mixed models to examine associations between donor age and both 1-year graft failure and primary graft dysfunction (PGD). The rate of 1-year graft failure was similar among recipients of lungs from donors age 18-64 years, but severely ill recipients (Lung Allocation Score [LAS] >47.7 or use of mechanical ventilation) of lungs from donors age 56-64 years had increased rates of 1-year graft failure (p-values for interaction = 0.04 and 0.02, respectively). Recipients of lungs from donors <18 and ≥65 years had increased rates of 1-year graft failure (adjusted hazard ratio [HR] 1.23, 95% CI 1.01-1.50 and adjusted HR 2.15, 95% CI 1.47-3.15, respectively). Donor age was not associated with the risk of PGD. In summary, the use of lungs from donors age 56 to 64 years may be safe for adult candidates without a high LAS and the use of lungs from pediatric donors is associated with a small increase in early graft failure.

Circulation and turnover of synaptic vesicle membrane in cultured fetal mammalian spinal cord neurons.

Intact neurons in cultures of fetal rodent spinal cord explants show stimulation-dependent uptake of horseradish peroxidase (HRP) into many small vesicles and occasional tubules and multivesicular bodies (MVB) at presynaptic terminals. Presynaptic terminals were allowed to take up HRP during 1 h of strychnine-enhanced stimulation of synaptic transmitter release and then "chased" in tracer-free medium either with strychnine or with 10 mM Mg++ which depresses transmitter release. Tracer-containing vesicles are lost from terminals under both chase conditions; the loss is more rapid (4-8 h) with strychnine than with 10 mM Mg++ (8-16 h). There is a parallel decrease in the numbers of labeled MVB's at terminals. Loss of tracer with 10 mM Mg++ does not appear to be due to the membrane rearrangements (exocytosis coupled to endocytosis) that presumably lead to initial tracer uptake; terminals exposed to HRP and Mg++ for up to 16 h show little tracer uptake into vesicles. Nor is the decrease likely to the due to loss of HRP enzyme activity; HRP is very stable in solution. During the chases there is a striking accumulation of HRP in perikarya that is far more extensive in cultures initially exposed to tracer with strychnine than 10 mM Mg++ regardless of chase conditions. Much of the tracer ends up in large dense bodies. These findings suggest that synaptic vesicle membrane turnover involves retrograde axonal transport of membrane to neuronal perikarya for further processing, including lysosomal degradation. The more rapid (4-8 h) loss of tracer-containing vesicles with strychnine may reflect vesicle membrane reutilization for exocytosis.

A light and electron microscope study of long-term organized cultures of rat dorsal root ganglia.

Dorsal root ganglia from fetal rats were explanted on collagen-coated coverslips and carried in Maximow double-coverslip assemblies for periods up to 3 months. These cultured ganglia were studied in the living state, in stained whole mounts, and in sections after OsO(4) fixation and Epon embedment. From the central cluster of nerve cell bodies, neurites emerge to form a rich network of fascicles which often reach the edge of the carrying coverslip. The neurons resemble their in vivo counterparts in nuclear and cytoplasmic content and organization; e.g., they appear as "light" or "dark" cells, depending on the amount of cytoplasmic neurofilaments. Satellite cells form a complete investment around the neuronal soma and are themselves everywhere covered by basement membrane. The neuron-satellite cell boundary is complicated by spinelike processes arising from the neuronal soma. Neuron size, myelinated fiber diameter, and internode length in the cultures do not reach the larger of the values known for ganglion and peripheral nerve in situ (30). Unmyelinated and myelinated nerve fibers and associated Schwann cells and endoneurial and perineurial components are organized into typical fascicles. The relationship of the Schwann cell and its single myelinated fiber or numerous unmyelinated fibers and the properties of myelin, such as lamellar spacing, mesaxons, Schmidt-Lanterman clefts, nodes of Ranvier, and protuberances, mimic the in vivo pattern. It is concluded that cultivation of fetal rat dorsal root ganglia by this technique fosters maturation and long-term maintenance of all the elements that comprise this cellular community in vivo (except vascular components) and, furthermore, allows these various components to relate faithfully to one another to produce an organotypic model of sensory ganglion tissue.

Effects of thallium salts on neuronal mitochondria in organotypic cord-ganglia-muscle combination cultures.

A functionally coupled organotypic complex of cultured dorsal root ganglia, spinal cord peripheral nerve, and muscle has been employed in an experimental approach to the investigation of the neurotoxic effects of thallium. Selected cultures, grown for up to 12 wk in vitro, were exposed to thallous salts for periods ranging up to 4 days. Cytopathic effects were first detected after 2 h of exposure with the appearance of considerably enlarged mitochondria in axons of peripheral nerve fibers. With time, the matrix space of these mitochondria became progressively swollen, transforming the organelle into an axonal vacuole bounded by the original outer mitochondrial membrane. Coalescence of adjacent axonal vacuoles produced massive internal axon compartments, the membranes of which were shown by electron microprobe mass spectrometry to have an affinity for thallium. Other axoplasmic components were displaced within a distended but intact axolemma. The resultant fiber swelling caused myelin retraction from nodes of Ranvier but no degeneration. Impulses could still propagate along the nerve fibers throughout the time course of the experiment. Comparable, but less severe changes were seen in dorsal root ganglion neurons and in central nerve fibers. Other cell types showed no mitochondrial change. It is uncertain how these findings relate to the neurotoxic effects of thallium in vivo, but a sensitivity of the nerve cell and especially its axon to thallous salts is indicated.

Development of specific sensory-evoked synaptic networks in fetal mouse cord-brainstem cultures.

Neurites of nerve growth factor-enhanced fetal mouse dorsal root ganglion cells can not only establish characteristic sensory synaptic network functions in dorsal regions of attached spinal cord explants, but some of the neurites may grow through the cord tissue in these cultures and make similar functional synaptic connections with specific types of "target" neurons in localized zones within nearby medulla explants.

Nerve growth factor attenuates neurotoxic effects of taxol on spinal cord-ganglion explants from fetal mice.

Most neurons in organotypic cultures of dorsal root ganglia from 13-day-old fetal mice require high concentrations of nerve growth factor for survival during the first week after explanation. These nerve growth factor-enhanced sensory neurons mature and innervate the dorsal regions of attached spinal cord tissue even after the removal of exogenous growth factor after 4 days. In cultures exposed for 4 days to nerve growth factor and taxol (a plant alkaloid that promotes the assembly of microtubules) and returned to medium without growth factor, greater than 95 percent of the ganglionic neurons degenerated and the spinal cord tissues were reduced almost to monolayers. In contrast, when the recovery medium was supplemented with nerve growth factor, the ganglionic neurons and dorsal (but not ventral) cord tissue survived remarkably well. Dorsal cord neurons do not normally require an input from dorsal root ganglia for long-term maintenance in vitro, but during and after taxol exposure they become dependent for survival and recovery on the presence of neurite projections from nerve growth-factor-enhanced dorsal root ganglia.

Ultrastructural studies of the dying-back process. VI. Examination of nerve fibers undergoing giant axonal degeneration in organotypic culture.

Organotypic tissue cultures, composed of structurally and functionally coupled explants of mouse spinal cord, dorsal root ganglia, and striated muscle, have been used to create a model of the distal (dying-back) axonopathy found in animals and humans with aliphatic hexacarbon neuropathy. Mature explants were treated with 50-650 micrograms/ml of the following hexacarbons dissolved in nutrient fluid: n-hexane, 2-hexanol, 2,5-hexanediol, methyl n-butyl ketone, 5-hydroxy-2-hexanone, 2,5-hexanedione (all neurotoxic), or 2,4-hexanedione (a non-neurotoxic diketone). High concentrations (400-650 micrograms/ml) induced pancytotoxic damage and necrosis of tissue within days, while the lower doses (50-100 micrograms/ml) induced no pathological changes over a period of several weeks. Continuous exposure of explants to 245-325 micrograms/ml (2.8 mM) of the neurotoxic hexacarbons caused specific pathological changes to develop in distal nerve fibers after three to six weeks. Initial changes seen in distal, nonterminal regions of myelinated fibers included: nodal elongation, axonal swellings on proximal-side paranodes, and paranodal myelin retraction. Prolonged treatment was associated with Wallerian-like degeneration of distal nerve fibers. Denuded paranodal swellings in more proximal regions of affected myelinated fibers adopted a more-normal size and underwent remyelination; this occurred during and after the course of treatment. Remyelination by lateral extension from adjacent Schwann cells was documented in living and fixed tissue. The observations confirm the spatial-temporal evolution of hexacarbon distal axonopathy previously suggested from comparable studies in vivo.

Conformationally restricted inhibitors of angiotensin converting enzyme: synthesis and computations.

A series of inhibitors of angiotensin converting enzyme (ACE, dipeptidyl carboxypeptidase, EC 3.4.15.1) is described which addresses certain conformational aspects of the enzyme-inhibitor interaction. In this study the alanylproline portion of the potent ACE inhibitor enalaprilat (2) is replaced by a series of monocyclic lactams containing the required recognition and binding elements. In order to more fully assess the lactam ring conformations and the key backbone angle psi as defined in 3 with respect to possible enzyme-bound conformations, a series of model lactams was investigated with use of molecular mechanics. The results point to a correlation between inhibitor potency (IC50) and the computed psi angle for the lowest energy conformation of the model compounds. Thus the psi angle as defined in 3 is an important determinant in the binding of inhibitors to ACE. The inhibition data in conjunction with the computational data have served to define a window of psi angles from 130 degrees to 170 degrees which seems to be acceptable to the ACE active site.

Morphological alterations in dorsal root ganglion neurons and supporting cells of organotypic mouse spinal cord-ganglion cultures exposed to taxol.

Explants of 14-day fetal mouse spinal cord with attached dorsal root ganglia, which had become differentiated over 2-3 weeks in culture, were exposed to 1-2 microM taxol for up to 6 days. The culture medium was supplemented with nerve growth factor (300 units/ml) during exposure to the drug. By 3-6 days in taxol, unusually numerous microtubules were seen in peripheral perikaryal and proximal neuritic regions of ganglion neurons. Microtubules also engirdled massive aggregations of pleomorphic vesicular/cisternal elements in many neurons. These aggregates were visible as unusual 'clear' spheroidal regions in the living cells, and were often as large as the nuclei. Some of the elements comprising these striking vesicular/cisternal accumulations appeared to be portions of disrupted Golgi complexes normally polarized around the cytocentrum, as well as hypertrophied smooth endoplasmic reticulum formations. In other neuronal areas, Golgi complexes and other organelles were altered or disrupted to lesser degrees. Ordered microtubular arrays occurred along endoplasmic reticulum cisternae both in neuron somata and neurites. Over time, a plethora of microtubules assembled throughout the perikarya in various orientations apparently unrelated to microtubule organizing centers. Unlike the effects of other plant alkaloids that interact with tubulin, there was no discernible increase in filaments, although their distribution appeared altered. Concentric ordered microtubular-macromolecular lamellated complexes were seen only in neurites. Neuronal nuclei were misshapen, often displaced, and displayed fine structure reminiscent of chromatolysis. Satellite and Schwann cells contained atypically abundant microtubules, abnormal cisternae, disrupted Golgi complexes, and increased lysosomes. Some nuclei displayed abnormal chromatin, and in rare cases even microtubules. We suggest that taxol alters the distribution, integrity, and/or organization of organelle systems in dorsal root ganglion cells by engendering unusually abundant microtubules in abnormal groupings and aberrant locations in these cells.

Presence of leucine-enkephalin in organotypic explants of fetal mouse spinal cord.

Spinal cord explants with attached dorsal root ganglia (DRGs), from 14-day fetal mice were fixed at 1-3 weeks in vitro and incubated for leucine-enkephalin (LE) immunoreactivity by the peroxidase-anti-peroxidase (PAP) immunohistochemical method. Results show long processes with labeled varicosities seen more often in dorsal regions of the cord explants. Stained punctate bodies and varicosities were often seen close to large cells in these cultures, whereas no label was detected in neuronal perikarya. A prominent laminar array of stained punctate bodies was noted in one cord explant, concentric with the perimeter of the explant. No LE label was detected in the neuritic outgrowths from the cord-DRG explants, whereas high levels of opiate receptors develop in these outgrowths, primarily on the DRG neurites, by 1-2 weeks in culture. The results indicate the presence of LE in explants of fetal mouse spinal cord with attached DRGs and offer an in vitro model system in which the onset and development of peptidergic neurons can be studied as they form functional cellular interrelationships with neurons bearing opioid and monoaminergic receptors in these organotypic cultures.

Selective innervation of target regions within fetal mouse spinal cord and medulla explants by isolated dorsal root ganglia in organotypic co-cultures.

Correlative electrophysiologic and cytologic analyses demonstrate that a significant group of neurons in isolated (NGF-enhanced) fetal mouse dorsal root ganglia (DRGs) can grow across a collagen substrate and selectively innervate specific dorsal horn and dorsal column nuclei regions in co-cultured explants of deafferented spinal cord and medulla. Neurites from this group of DRG cells from connections in CNS target zones that generate characteristic primary afferent network responses to sensory stimuli, as observed in cultures of spinal cord with attached DRGs. Systematic microelectrode stimulus-mapping tests revealed that many DRG neurites were preferentially distributed in sensory target zones of co-cultured cord and medulla explants and that few collaterals of these DRG neurons were present in neighboring inappropriate regions, especially in the ventral cord. Another group of DRG neurons appears to be responsible for the less prominent, but clear-cut, innervation that developed in some of the co-cultured ventral cord explants. Fetal DRGs were also able to establish characteristic primary afferent dorsal horn or dorsal column nuclei networks when introduced into cultures of deafferented spinal cord and medulla that had been explanted alone for 1-3 weeks prior to introduction of the DRGs. These experiments demonstrate that CNS target neurons remain receptive to DRG innervation even after 1-3 weeks of maturation in vitro. Our electrophysiologic and cytologic analyses of DRG and CNS explants in organotypic co-cultures provide the first systematic attempt to establish conditions under which preferential neuritic growth to and functional innervation of specific CNS target tissues can occur in vitro. This model system should facilitate analyses of mechanisms underlying development, as well as regeneration, of specific synaptic connections in the CNS.

Preferential growth of neurites from isolated fetal mouse dorsal root ganglia in relation to specific regions of co-cultured spinal cord explants.

Clusters of dorsal root ganglia (DRGs) from 13- to 14-day fetal mice were co-cultured with specific fragments of deafferented spinal cord (0.5-1 mm apart) on collagen-coated coverslips in Maximow slide chambers. Nerve growth factor (NGF) was added to the culture medium (1000 biological units/ml, at explantation) to ensure optimal survival and growth of a large fraction of the fetal DRG neurons. Sequential microscopic observations of the living cultures and cytologic studies after silver impregnation demonstrate that many neurites from isolated DRGs can invade dorsal (DC) regions of co-cultured spinal cord explants, whereas they are deflected from ventral cord (VC) tissue and its neuritic-glial outgrowth. Furthermore, some DRG neurites may become redirected towards distant DC target explants even after long circuitous detours around proximally arrayed VC explants. DRG neurites also show remarkably sharp projections to DC tissue and more complete avoidance of adjacent VC tissue when the DRG neurites approach a suitably arranged DC-VC-DC interface, e.g. forming de novo 'dorsal roots' at the end of a longitudinal strip of whole spinal cord. These experiments suggest that DC-VC boundaries may be particularly effective in guiding DRG neurites to specific regions of the CNS. The present studies of co-cultured fetal mouse DRG and spinal cord explants provide the first demonstration of preferential neuritic growth in vitro in relation to specific CNS target tissues.

Cyclic AMP or forskolin rapidly attenuates the depressant effects of opioids on sensory-evoked dorsal-horn responses in mouse spinal cord-ganglion explants.

Exposure of fetal mouse spinal cord-ganglion explants to morphine (greater than 0.1 microM) results in naloxone-reversible, dose-dependent depression of sensory-evoked dorsal-horn synaptic-network responses within a few minutes. After chronic opiate exposure (1 microM) for 2-3 days, these dorsal cord responses recover and can then occur even in greater than 10 microM morphine. In the present study, when naive explants were treated with forskolin (10-50 microM)--a selective activate activator of cyclase (AC)--for 10-30 min prior to and during exposure to morphine (0.1-0.3 microM) or D-Ala2-D-Leu5-enkephalin (0.03-0.1 microM), the usual opioid depressant effects on dorsal-horn responses generally failed to occur (10-30 min tests). Dibutyryl cyclic AMP (10 microM) or the more lipid-soluble analog, dioctanoyl cyclic AMP (0.1 mM), produced a similar degree of subsensitivity to opiates as 10 microM forskolin. With high levels of forskolin (50 microM), even concentrations of morphine up to 1-10 microM were far less effective in depressing cord responses. These effects of exogenous cAMP analogs and forskolin on cord-ganglion explants are probably both mediated by increases in intracellular cAMP. The marked decrease in opioid sensitivity of cAMP or forskolin-treated cord-ganglion explants provides significant electrophysiologic data compatible with the hypothesis that neurons may develop tolerance and/or dependence during chronic opioid exposure by a compensatory enhancement of their AC/cAMP system following initial opioid depression of AC activity. Previous evidence relied primarily on behavioral tests and biochemical analyses of cell cultures. It will be of interest to determine if dorsal-horn tissues of cord-ganglion explants do, in fact, develop increased AC/cAMP levels as they express physiologic signs of tolerance during chronic exposure to opioids.

Specific neuritic pathways and arborizations formed by fetal mouse dorsal root ganglion cells within organized spinal cord explants in culture: a peroxidase-labeling study.

Extracellular microiontophoretic injections of horseradish peroxidase (HRP) into NGF-enhanced fetal mouse dorsal root ganglia (DRGs) produced an anterograde solid Golgi-like labeling of DRG neurites and their terminal arborizations within co-cultured spinal cord explants. In cultures of spinal cord transverse cross-sections with attached DRGs, the large NGF-enhanced DRGs remained in close proximity to the cord, often adjacent to both dorsal and ventral cord regions. Despite this, nearly all DRG neurites that entered the cord did so via dorsal root fascicles. They branched and ramified extensively within the dorsal region, taking on a wavy or kinky course and showed various types of arborizations. The density of cord innervation was much lower when isolated DRGs and cord explants were co-cultured 0.5-1 mm apart. Although fewer entering DRG fibers were labeled by our HRP injections the same qualitative growth and arborization patterns were seen within dorsal and ventral cord regions as in explants of cord with attached DRGs. When the facing edge contained both dorsal and ventral tissues, HRP-labeled DRG fibers entered dorsal regions selectively. DRG fibers readily entered, ramified and arborized within isolated strips of dorsal cord, whereas they sharply avoided isolated ventral cord explants. The avoidance of ventral cord cannot simply be due to the paucity of specific synaptic targets within the tissue, for larger numbers of DRG fibers entered completely inappropriate CNS target tissues, e.g. superior colliculus explants--though they did not ramify or arborize to any degree comparable to that seen within dorsal cord regions.

Neurites from mouse retina and dorsal root ganglion explants show specific behavior within co-cultured tectum or spinal cord.

We have utilized extracellular microiontophoretic injections of horseradish peroxidase into fetal mouse retinal explants to label retinal ganglion cell axons innervating co-cultured tectal explants in a solid Golgi-like manner. Using dorsal root ganglia-tectum and retina-spinal cord co-cultures as controls, our results indicate that retinal neurites show selective growth and arborizations within their appropriate tectal, target tissue. Retinotectal explant co-cultures may be a useful model system for studying aspects of neuronal specificity.

The twitcher mouse: myelinogenesis in organotypic culture.

Myelinogenesis was followed in organotypic cultures of the spinal cord of the neurological mutant mouse, the Twitcher. As a clinically, pathologically and biochemically equivalent model of Krabbe disease this mutant is an important tool for investigating the nervous system. Normal initiation and development of myelination was observed. At 35 days in vitro (DIV) the Twitcher cultures exhibited blisters attached to the intact myelin sheath and bubbling of myelin suggestive of myelin breakdown. Myelin degeneration progressed thereafter. The Twitcher spinal cord survived in culture for more than two months, a period much longer than the life span of affected mice. In order to correlate pathological and biochemical changes, the activity of UDP-galactose:ceramide galactosyltransferase was quantitated in normal and Twitcher cultures. In both the Twitcher and the control groups the galactosyltransferase activity rapidly increased up to 20-25 DIV and then declined. The galactosyltransferase activity of the Twitcher tended to be lower than the controls even during the early myelination period. At 35 DIV the activity in the Twitcher was definitely lower than the controls, and at 52 DIV it was nearly negligible. The galactosyltransferase activity therefore correlated well with the morphologically observed early normal myelination and subsequent myelin degeneration.

Microtubule arrays in taxol-treated mouse dorsal root ganglion-spinal cord cultures.

Exposure of organotypic dorsal root ganglion-spinal cord cultures to taxol, a potent microtubule promoting and stabilizing agent, results in an unusual abundance of microtubules in neurons, and the presence of microtubule-endoplasmic reticulum arrays in their perikarya and processes. Ordered concentric arrays of microtubules alternating with marcromolecular material are observed in dorsal root ganglion neurites. Short linear structures are discernible between some microtubules and the macromolecular material, as well as between microtubules and endoplasmic reticulum cisternae. Analyses of such unusual microtubule arrays in taxol-treated cultures may provide valuable insights into tubulin-related systems in neurons, as well as in other cells.

Myelination in organotypic cultures of sympathetic ganglia.

Explants of mouse superior cervical ganglion (SCG), co-cultured with dorsal spinal cord, were grown for up to 4 weeks in vitro. In such cultures, scattered internodes of peripheral nervous system (PNS) myelin were observed, apparently associated with SCG neurites. Although rare, the incidence of PNS myelination in this system might merit further experimentation to provide a model facilitating the evaluation of postganglionic sympathetic myelination, which in vivo may be both extensive and morphologically unusual.