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.

X-ray microprobe of orbital alignment in strong-field ionized atoms.

We have developed a synchrotron-based, time-resolved x-ray microprobe to investigate optical strong-field processes at intermediate intensities (10(14) - 10(15) W/cm2). This quantum-state specific probe has enabled the direct observation of orbital alignment in the residual ion produced by strong-field ionization of krypton atoms via resonant, polarized x-ray absorption. We found strong alignment to persist for a period long compared to the spin-orbit coupling time scale (6.2 fs). The observed degree of alignment can be explained by models that incorporate spin-orbit coupling. The methodology is applicable to a wide range of problems.

Differential expression of creatine kinase and phosphoglycerate mutase isozymes during development in aneural and innervated human muscle culture.

Several enzymes that occur in multimolecular forms undergo transitions during myogenesis. Studies of such developmentally regulated isozymes (e.g. creatine kinase) indicate that muscle cells, cultured in the absence of neural tissue never develop fully mature isozyme patterns, but continue to express large amounts of 'housekeeping' isozymes that are characteristically present in fetal muscle. We studied two developmentally controlled isozymes, creatine kinase (CK) and phosphoglycerate mutase (PGAM) in normal human muscle, both aneurally cultured and co-cultured with fetal mouse spinal cord complex. Innervated cultures attain a greater degree of maturity than non-innervated cultures, as revealed by light and electron microscopy, showing well-developed sarcomeres and motor endplates after several weeks in vitro. During early stages of muscle regeneration in co-culture, characteristic fetal isozyme patterns of CK-BB and PGAM-BB activity predominate, as in aneural cultures. The muscle-specific isozymes (CK-MM; PGAM-MM) begin to appear as the muscle differentiates, and after 2-3 months in co-culture only, virtually all enzyme activity is due to the muscle-specific forms of CK and PGAM, as is normally observed in mature skeletal muscle in vivo.

Nerve growth factor regulates the action potential duration of mature sensory neurons.

The effect of nerve growth factor (NGF) on the action potential of sensory ganglion neurons was investigated in long-term organotypic cultures of embryonic mouse dorsal root ganglia grown isolated or attached to spinal cord explants. The present study demonstrates that NGF regulates a specific bioelectric property of these neurons--the duration of the Ca2+ component of the somatic action potential--at mature stages when they no longer require NGF for survival. Prolonged culture of fetal mouse dorsal root ganglion neurons with relatively low levels of NGF shortens the duration of the action potential. Furthermore, addition or withdrawal of NGF in mature cultures results, within several days, in shorter or longer action potential durations, respectively. Exposure to anti-NGF antiserum accelerates the onset of the longer-lasting action potentials elicited by simple withdrawal of NGF. This plastic response of sensory neurons to NGF may be important in regulating their physiological properties and/or their response to injury.

Duchenne dystrophic muscle develops lesions in long-term coculture with mouse spinal cord.

When strips of human skeletal muscle from biopsies of normal children and donors with Duchenne muscular dystrophy (DMD) are explanted in organotypic coculture with fetal mouse spinal cord, many regenerating muscle fibers develop, become innervated, and maintain a remarkable degree of mature structure and function for more than 3-6 months in vitro. Sequential light microscopy in correlation with electron-microscopic and electrophysiologic analyses showed that despite cross-species innervation, these human muscle fibers develop stable cross-striations, peripherally positioned myonuclei, and mature, functional motor endplates. Of special interest is the onset of significant progressive abnormalities, e.g., unusual focal myofibrillar lesions, in substantial numbers of innervated mature DMD muscle fibers after 2-4 months in culture. The focal myofibrillar lesions were not detected in normal muscle fibers maintained as long as 6 months in coculture, nor are they comparable to the generalized loss of cross-striations observed in muscle atrophy following in vitro denervation of mature DMD fibers.

Chemical neurotoxicity: detection and analysis in organotypic cultures of sensory and motor systems.

Screening of chemical substances for human neurotoxic (and therapeutic) properties may be carried out with the aid of organotypic tissue cultures composed of foetal explants of mouse sensory and neuromuscular tissues that develop in vitro their characteristic cytoarchitectural and functional organization. Supporting this statement is a wealth of studies describing a range of specific, chemically-induced responses in organotypic neural cultures that parallel changes induced in the nervous system of humans and animals.

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.

Antagonist-induced opiate receptor upregulation in cultures of fetal mouse spinal cord-ganglion explants.

Chronic exposure of fetal mouse spinal cord-ganglion explants to the opioid antagonist naloxone (10 microM, 7 days) produced a pronounced upregulation of mu opioid receptors. The antagonist action was stereospecific, as it was produced by (-)-, but not by (+)-naloxone, and was dose-dependent. Half-maximal naloxone-induced receptor upregulation occurred after two days; receptor density was maximal at 5 days. Exposure of the explant cultures to naloxone (10 microM) in the presence of the protein synthesis inhibitor, cycloheximide (1 microM; a concentration which blocks greater than 90% protein synthesis) resulted in receptor density changes that were similar to those observed in cultures exposed to naloxone alone. This finding suggests that antagonist-induced opiate receptor upregulation does not require the synthesis of new receptor molecules.

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.

Taxol effects on glia in organotypic mouse spinal cord-DRG cultures.

Exposure of organotypic mouse spinal cord-dorsal root ganglion cultures to 1-2 microM taxol for up to 6 days results in a remarkable increase in cytoplasmic microtubules in differentiated oligodendroglia. In contrast, there is a notably smaller change in the microtubule complement of astroglia. This suggests that the regulatory and/or synthetic systems for tubulin in these glial types may differ, and that oligodendroglia appear more responsive to modulation in the levels of tubulin subunits as a result of the action of taxol.

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.

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.

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.

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.

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.

Development of cross-tolerance to 5-hydroxytryptamine in organotypic cultures of mouse spinal cord-ganglia during chronic exposure to morphine.

Exposure of organotypic explants of mouse spinal cord with attached dorsal root ganglia (DRGs) to low concentrations (approximately 10nM) of 5-hydroxytryptamine (5-HT) markedly depressed sensory-evoked dorsal-horn network responses, resembling the acute effects of opioids in these cultures. Attenuation of cord responses by 5-HT was not prevented by exposure to the 5-HT antagonists, methysergide and cyproheptadiene, nor to the opiate antagonist, naloxone. Explants that had become tolerant to morphine after chronic exposure (1 microM) for greater than 2 days often developed cross-tolerance to 5-HT. Acute exposure of morphine-tolerant explants to naloxone (1 microM) further attenuated the effects of 5-HT so that the minimum depressant levels of 5-HT were often increased up to 30-fold. Increasing the extra-cellular Ca++ concentration (to 5 mM) and/or introduction of 4-aminopyridine markedly antagonized the depressant effects of 5-HT on DRG-evoked cord responses, so that 5-HT levels comparable to those used on morphine-tolerant explants were required to depress naive explants. These depressant effects of 5-HT on cord-DRG explants are consonant with antinociceptive actions of 5-HT administered to dorsal cord in situ. Our data suggest that 5-HT may block neuronal components of dorsal horn networks at similar regions to those that are depressed by opiates, i.g. presynaptic DRG nerve terminals where abundant opiate receptors are located. The marked attenuation of the depressant effects of both 5-HT and opiates on cord-DRG explants by high Ca++ raises the possibility that cross-tolerance to 5-HT in morphine-tolerant explants may result from the same neuronal alterations that render dorsal-horn networks tolerant to opiates. Furthermore, the increased degree of cross-tolerant cultures may be an expression of opiate dependence.

Exposure to 4-aminopyridine prevents depressant effects of opiates on sensory-evoked dorsal-horn network responses in spinal cord cultures.

The depressant effects of morphine (0.1-1 microM) on sensory-evoked dorsal-horn network responses in explants of mouse spinal cord with attached dorsal root ganglia (DRGs) were rapidly restored after addition of 4-aminopyridine (4-AP; 0.1 mM) and major components of these cord responses were stably maintained in the presence of the opiate. Moreover, prior exposure of cord-DRG explants to 0.1 mM 4-AP prevented the depressant effects of 0.1 microM morphine on DRG-evoked dorsal-horn responses, and the effects of 1-10 microM morphine were at least partly antagonized. Increased Ca++ levels (5 microM) attenuated the depression of dorsal horn responses by 1-10 micro M morphine and these effects of Ca++ were greatly enhanced in the presence of 4-AP--in some cultures, concentrations of morphine as high as 100 micro M were strongly antagonized during test periods up to 2 hours. Receptor assays showed that 0.1 mM 4-AP +/- 5 mM Ca++ had no effect on stereospecific opiate binding, indicating that the antagonist actions of these agents in our cultures do not occur at the level of the opiate receptor. The relevance of our in vitro studies of 4-AP antagonism of opiate-depressant effects on sensory-evoked dorsal-horn network responses for analyses of problems in opiate analgesia has been strengthened by a recent report demonstrating that 4-AP does, in fact, reverse morphine analgesia in rats, as determined by tail flick tests.

Phosphorus-containing inhibitors of angiotensin-converting enzyme.

Several phosphonamides, phosphoramides, and phosphates having the general structure R-Y-P(O)(OH)-X-CH(CH3)-CO-Pro have been synthesized and tested for inhibition of angiotensin-converting enzyme (dipeptidyl carboxypeptidase; peptidyl-dipeptide hydrolase, EC 3.4.15.1). Inhibition was found to depend on the nature of R, Y, and X such that the maximal effect was observed when X = NH, Y = CH2, and R = phi CH2 (50% inhibition at 7 nM). Substitution of CH2 or O at X and O at Y produced significantly less potent inhibitors. Groups shorter or longer than R = phi CH2 led to less active inhibitors, presumably due to nonoptimal interaction of the side chain with the S1 subsite.