Traumatic brain injury results in unique microglial and astrocyte transcriptomes enriched for type I interferon response.

BACKGROUND: Traumatic brain injury (TBI) is a leading cause of death and disability that lacks neuroprotective therapies. Following a TBI, secondary injury response pathways are activated and contribute to ongoing neurodegeneration. Microglia and astrocytes are critical neuroimmune modulators with early and persistent reactivity following a TBI. Although histologic glial reactivity is well established, a precise understanding of microglia and astrocyte function following trauma remains unknown. METHODS: Adult male C57BL/6J mice underwent either fluid percussion or sham injury. RNA sequencing of concurrently isolated microglia and astrocytes was conducted 7 days post-injury to evaluate cell-type-specific transcriptional responses to TBI. Dual in situ hybridization and immunofluorescence were used to validate the TBI-induced gene expression changes in microglia and astrocytes and to identify spatial orientation of cells expressing these genes. Comparative analysis was performed between our glial transcriptomes and those from prior reports in mild TBI and other neurologic diseases to determine if severe TBI induces unique states of microglial and astrocyte activation. RESULTS: Our findings revealed sustained, lineage-specific transcriptional changes in both microglia and astrocytes, with microglia showing a greater transcriptional response than astrocytes at this subacute time point. Microglia and astrocytes showed overlapping enrichment for genes related to type I interferon signaling and MHC class I antigen presentation. The microglia and astrocyte transcriptional response to severe TBI was distinct from prior reports in mild TBI and other neurodegenerative and neuroinflammatory diseases. CONCLUSION: Concurrent lineage-specific analysis revealed novel TBI-specific transcriptional changes; these findings highlight the importance of cell-type-specific analysis of glial reactivity following TBI and may assist with the identification of novel, targeted therapies.

Cerebrospinal Fluid Markers of Macrophage and Lymphocyte Activation After Traumatic Brain Injury in Children.

OBJECTIVES: The magnitude and role of the cellular immune response following pediatric traumatic brain injury remains unknown. We tested the hypothesis that macrophage/microglia and T-cell activation occurs following pediatric traumatic brain injury by measuring cerebrospinal fluid levels of soluble cluster of differentiation 163 and ferritin and soluble interleukin-2 receptor α, respectively, and determined whether these biomarkers were associated with relevant clinical variables and outcome. DESIGN: Retrospective analysis of samples from an established, single-center cerebrospinal fluid bank. SETTING: PICU in a tertiary children's hospital. PATIENTS: Sixty-six pediatric patients after severe traumatic brain injury (Glasgow Coma Scale score < 8) who were 1 month to 16 years old and 17 control patients who were 1 month to 14 years old. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Cerebrospinal fluid levels of soluble cluster of differentiation 163, ferritin, and soluble interleukin-2 receptor α were determined by enzyme-linked immunosorbent assay at two time points (t1 = 17 ± 10 hr; t2 = 72 ± 15 hr) for each traumatic brain injury patient. Cerebrospinal fluid levels of soluble cluster of differentiation 163, ferritin, and soluble interleukin-2 receptor α after traumatic brain injury were compared with controls and analyzed for associations with age, patient sex, initial Glasgow Coma Scale score, diagnosis of abusive head trauma, the presence of hemorrhage on CT scan, and Glasgow Outcome Scale score. Cerebrospinal fluid level of soluble cluster of differentiation 163 was increased in traumatic brain injury patients at t2 versus t1 and controls (median, 95.4 ng/mL [interquartile range, 21.8-134.0 ng/mL] vs 31.0 ng/mL [5.7-77.7 ng/mL] and 27.8 ng/mL [19.1-43.1 ng/mL], respectively; p < 0.05). Cerebrospinal fluid level of ferritin was increased in traumatic brain injury patients at t2 and t1 versus controls (8.3 ng/mL [<7.5-19.8 ng/mL] and 8.9 ng/mL [<7.5-26.7 ng/mL] vs <7.5 ng/mL below lower limit of detection, respectively; p < 0.05). Cerebrospinal fluid levels of soluble interleukin-2 receptor α in traumatic brain injury patients at t2 and t1 were not different versus controls. Multivariate regression revealed associations between high ferritin and age 4 years or younger, lower Glasgow Coma Scale score, abusive head trauma, and unfavorable Glasgow Outcome Scale score. CONCLUSIONS: Children with traumatic brain injury demonstrate evidence for macrophage activation after traumatic brain injury, and in terms of cerebrospinal fluid ferritin, this appears more prominent with young age, initial injury severity, abusive head trauma, and unfavorable outcome. Further study is needed to determine whether biomarkers of macrophage activation may be used to discriminate between aberrant and adaptive immune responses and whether inflammation represents a therapeutic target after traumatic brain injury.

NOX2 protects against progressive lung injury and multiple organ dysfunction syndrome.

Systemic inflammatory response syndrome (SIRS) is a common clinical condition in patients in intensive care units that can lead to complications, including multiple organ dysfunction syndrome (MODS). MODS carries a high mortality rate, and it is unclear why some patients resolve SIRS, whereas others develop MODS. Although oxidant stress has been implicated in the development of MODS, several recent studies have demonstrated a requirement for NADPH oxidase 2 (NOX2)-derived oxidants in limiting inflammation. We recently demonstrated that NOX2 protects against lung injury and mortality in a murine model of SIRS. In the present study, we investigated the role of NOX2-derived oxidants in the progression from SIRS to MODS. Using a murine model of sterile systemic inflammation, we observed significantly greater illness and subacute mortality in gp91(phox-/y) (NOX2-deficient) mice compared with wild-type mice. Cellular analysis revealed continued neutrophil recruitment to the peritoneum and lungs of the NOX2-deficient mice and altered activation states of both neutrophils and macrophages. Histological examination showed multiple organ pathology indicative of MODS in the NOX2-deficient mice, and several inflammatory cytokines were elevated in lungs of the NOX2-deficient mice. Overall, these data suggest that NOX2 function protects against the development of MODS and is required for normal resolution of systemic inflammation.

Neuroprotective effects of naltrexone in a mouse model of post-traumatic seizures.

Traumatic Brain Injury (TBI) induces neuroinflammatory response that can initiate epileptogenesis, which develops into epilepsy. Recently, we identified anti-convulsive effects of naltrexone, a mu-opioid receptor (MOR) antagonist, used to treat drug addiction. While blocking opioid receptors can reduce inflammation, it is unclear if post-TBI seizures can be prevented by blocking MORs. Here, we tested if naltrexone prevents neuroinflammation and/or seizures post-TBI. TBI was induced by a modified Marmarou Weight-Drop (WD) method on 4-week-old C57BL/6J male mice. Mice were placed in two groups: non-telemetry assessing the acute effects or in telemetry monitoring for interictal events and spontaneous seizures both following TBI and naltrexone. Molecular, histological and neuroimaging techniques were used to evaluate neuroinflammation, neurodegeneration and fiber track integrity at 8 days and 3 months post-TBI. Peripheral immune responses were assessed through serum chemokine/cytokine measurements. Our results show an increase in MOR expression, nitro-oxidative stress, mRNA expression of inflammatory cytokines, microgliosis, neurodegeneration, and white matter damage in the neocortex of TBI mice. Video-EEG revealed increased interictal events in TBI mice, with 71% mice developing post-traumatic seizures (PTS). Naltrexone treatment ameliorated neuroinflammation, neurodegeneration, reduced interictal events and prevented seizures in all TBI mice, which makes naltrexone a promising candidate against PTS, TBI-associated neuroinflammation and epileptogenesis in a WD model of TBI.

Selective neuroimmune modulation by type I interferon drives neuropathology and neurologic dysfunction following traumatic brain injury.

Accumulating evidence suggests that type I interferon (IFN-I) signaling is a key contributor to immune cell-mediated neuropathology in neurodegenerative diseases. Recently, we demonstrated a robust upregulation of type I interferon-stimulated genes in microglia and astrocytes following experimental traumatic brain injury (TBI). The specific molecular and cellular mechanisms by which IFN-I signaling impacts the neuroimmune response and neuropathology following TBI remains unknown. Using the lateral fluid percussion injury model (FPI) in adult male mice, we demonstrated that IFN α/ß receptor (IFNAR) deficiency resulted in selective and sustained blockade of type I interferon-stimulated genes following TBI as well as decreased microgliosis and monocyte infiltration. Molecular alteration of reactive microglia also occurred with diminished expression of genes needed for MHC class I antigen processing and presentation following TBI. This was associated with decreased accumulation of cytotoxic T cells in the brain. The IFNAR-dependent modulation of the neuroimmune response was accompanied by protection from secondary neuronal death, white matter disruption, and neurobehavioral dysfunction. These data support further efforts to leverage the IFN-I pathway for novel, targeted therapy of TBI.

Selective neuroimmune modulation by type I interferon drives neuropathology and neurologic dysfunction following traumatic brain injury.

Accumulating evidence suggests that type I interferon (IFN-I) signaling is a key contributor to immune cell-mediated neuropathology in neurodegenerative diseases. Recently, we demonstrated a robust upregulation of type I interferon-stimulated genes in microglia and astrocytes following experimental traumatic brain injury (TBI). The specific molecular and cellular mechanisms by which IFN-I signaling impacts the neuroimmune response and neuropathology following TBI remains unknown. Using the lateral fluid percussion injury model (FPI) in adult male mice, we demonstrated that IFN α/ß receptor (IFNAR) deficiency resulted in selective and sustained blockade of type I interferon-stimulated genes following TBI as well as decreased microgliosis and monocyte infiltration. Phenotypic alteration of reactive microglia also occurred with diminished expression of molecules needed for MHC class I antigen processing and presentation following TBI. This was associated with decreased accumulation of cytotoxic T cells in the brain. The IFNAR-dependent modulation of the neuroimmune response was accompanied by protection from secondary neuronal death, white matter disruption, and neurobehavioral dysfunction. These data support further efforts to leverage the IFN-I pathway for novel, targeted therapy of TBI.

A Mouse Model for Juvenile, Lateral Fluid Percussion Brain Injury Reveals Sex-Dependent Differences in Neuroinflammation and Functional Recovery.

Traumatic brain injury (TBI) is a leading cause of death and disability that lacks targeted therapies. Successful translation of promising neuroprotective therapies will likely require more precise identification of target populations through greater study of crucial biological factors like age and sex. A growing body of work supports the impact of these factors on response to and recovery from TBI. However, age and sex are understudied in TBI animal models. The first aim of this study was to demonstrate the feasibility of lateral fluid percussion injury (FPI) in juvenile mice as a model of pediatric TBI. Subsequently, we were interested in examining the impact of young age and sex on TBI outcome. After adapting the lateral FPI model to 21-day-old male and female mice, we characterized the molecular, histological, and functional outcomes. Whereas similar tissue injury was observed in male and female juvenile mice exposed to TBI, we observed differences in neuroinflammation and neurobehavioral function. Overall, our findings revealed less acute inflammatory cytokine expression, greater subacute microglial/macrophage accumulation, and greater neurological recovery in juvenile male mice after TBI. Given that ongoing brain development may affect progression of and recovery from TBI, juvenile models are of critical importance. The sex-dependent differences we discovered after FPI support the necessity of also including this biological variable in future TBI studies. Understanding the mechanisms underlying age- and sex-dependent differences may result in the discovery of novel therapeutic targets for TBI.

Sex Does Not Influence Visual Outcomes After Blast-Mediated Traumatic Brain Injury but IL-1 Pathway Mutations Confer Partial Rescue.

Purpose: In a mouse model of blast-mediated traumatic brain injury (bTBI), interleukin-1 (IL-1)-pathway components were tested as potential therapeutic targets for bTBI-mediated retinal ganglion cell (RGC) dysfunction. Sex was also evaluated as a variable for RGC outcomes post-bTBI. Methods: Male and female mice with null mutations in genes encoding IL-1α, IL-1ß, or IL-1RI were compared to C57BL/6J wild-type (WT) mice after exposure to three 20-psi blast waves given at an interblast interval of 1 hour or to mice receiving sham injury. To determine if genetic blockade of IL-1α, IL-1ß, or IL-1RI could prevent damage to RGCs, the function and structure of these cells were evaluated by pattern electroretinogram and optical coherence tomography, respectively, 5 weeks following blast or sham exposure. RGC survival was also quantitatively assessed via immunohistochemical staining of BRN3A at the completion of the study. Results: Our results showed that male and female WT mice had a similar response to blast-induced retinal injury. Generally, constitutive deletion of IL-1α, IL-1ß, or IL-1RI did not provide full protection from the effects of bTBI on visual outcomes; however, injured WT mice had significantly worse visual outcomes compared to the injured genetic knockout mice. Conclusions: Sex does not affect RGC outcomes after bTBI. The genetic studies suggest that deletion of these IL-1 pathway components confers some protection, but global deletion from birth did not result in a complete rescue.

Modulation of Post-Traumatic Immune Response Using the IL-1 Receptor Antagonist Anakinra for Improved Visual Outcomes.

The purpose of this study was to characterize acute changes in inflammatory pathways in the mouse eye after blast-mediated traumatic brain injury (bTBI) and to determine whether modulation of these pathways could protect the structure and function of retinal ganglion cells (RGC). The bTBI was induced in C57BL/6J male mice by exposure to three 20 psi blast waves directed toward the head with the body shielded, with an inter-blast interval of one hour. Acute cytokine expression in retinal tissue was measured through reverse transcription-quantitative polymerase chain reaction (RT-qPCR) four hours post-blast. Increased retinal expression of interleukin (lL)-1ß, IL-1α, IL-6, and tumor necrosis factor (TNF)α was observed in bTBI mice exposed to blast when compared with shams, which was associated with activation of microglia and macroglia reactivity, assessed via immunohistochemistry with ionized calcium binding adaptor molecule 1 and glial fibrillary acidic protein, respectively, one week post-blast. Blockade of the IL-1 pathway was accomplished using anakinra, an IL-1RI antagonist, administered intra-peritoneally for one week before injury and continuing for three weeks post-injury. Retinal function and RGC layer thickness were evaluated four weeks post-injury using pattern electroretinogram (PERG) and optical coherence tomography (OCT), respectively. After bTBI, anakinra treatment resulted in a preservation of RGC function and RGC structure when compared with saline treated bTBI mice. Optic nerve integrity analysis demonstrated a trend of decreased damage suggesting that IL-1 blockade also prevents axonal damage after blast. Blast exposure results in increased retinal inflammation including upregulation of pro-inflammatory cytokines and activation of resident microglia and macroglia. This may explain partially the RGC loss we observed in this model, as blockade of the acute inflammatory response after injury with the IL-1R1 antagonist anakinra resulted in preservation of RGC function and RGC layer thickness.

Combined Blockade of Interleukin-1α and -1ß Signaling Protects Mice from Cognitive Dysfunction after Traumatic Brain Injury.

Diffuse activation of interleukin-1 inflammatory cytokine signaling after traumatic brain injury (TBI) elicits progressive neurodegeneration and neuropsychiatric dysfunction, and thus represents a potential opportunity for therapeutic intervention. Although interleukin (IL)-1α and IL-1ß both activate the common type 1 IL-1 receptor (IL-1RI), they manifest distinct injury-specific roles in some models of neurodegeneration. Despite its potential relevance to treating patients with TBI, however, the individual contributions of IL-1α and IL-1ß to TBI-pathology have not been previously investigated. To address this need, we applied genetic and pharmacologic approaches in mice to dissect the individual contributions of IL-1α, IL-ß, and IL-1RI signaling to the pathophysiology of fluid percussion-mediated TBI, a model of mixed focal and diffuse TBI. IL-1RI ablation conferred a greater protective effect on brain cytokine expression and cognitive function after TBI than did individual IL-1α or IL-1ß ablation. This protective effect was recapitulated by treatment with the drug anakinra, a recombinant naturally occurring IL-1RI antagonist. Our data thus suggest that broad targeting of IL-1RI signaling is more likely to reduce neuroinflammation and preserve cognitive function after TBI than are approaches that individually target IL-1α or IL-1ß signaling.

Acute vitreoretinal trauma and inflammation after traumatic brain injury in mice.

Objective: Limited attention has been given to ocular injuries associated with traumatic brain injury (TBI). The retina is an extension of the central nervous system and evaluation of ocular damage may offer a less-invasive approach to gauge TBI severity and response to treatment. We aim to characterize acute changes in the mouse eye after exposure to two different models of TBI to assess the utility of eye damage as a surrogate to brain injury. Methods: A model of blast TBI (bTBI) using a shock tube was compared to a lateral fluid percussion injury model (LFPI) using fluid pressure applied directly to the brain. Whole eyes were collected from mice 3 days post LFPI and 24 days post bTBI and were evaluated histologically using a hematoxylin and eosin stain. Results: bTBI mice showed evidence of vitreous detachment in the posterior chamber in addition to vitreous hemorrhage with inflammatory cells. Subretinal hemorrhage, photoreceptor degeneration, and decreased cellularity in the retinal ganglion cell layer was also seen in bTBI mice. In contrast, eyes of LFPI mice showed evidence of anterior uveitis and subcapsular cataracts. Interpretation: We demonstrated that variations in the type of TBI can result in drastically different phenotypic changes within the eye. As such, molecular and phenotypic changes in the eye following TBI may provide valuable information regarding the mechanism, severity, and ongoing pathophysiology of brain injury. Because vitreous samples are easily obtained, molecular changes within the eye could be utilized as biomarkers of TBI in human patients.

2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine inhibit TNF-α and CXCL10 production from activated primary murine microglia via A2A receptors.

BACKGROUND: Some cells, tissues and organs release 2',3'-cAMP (a positional isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'-AMP plus 3'-AMP and convert these AMPs to adenosine (called the extracellular 2',3'-cAMP-adenosine pathway). Recent studies show that microglia have an extracellular 2',3'-cAMP-adenosine pathway. The goal of the present study was to investigate whether the extracellular 2',3'-cAMP-adenosine pathway could have functional consequences on the production of cytokines/chemokines by activated microglia. METHODS: Experiments were conducted in cultures of primary murine microglia. In the first experiment, the effect of 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine on LPS-induced TNF-α and CXCL10 production was determined. In the next experiment, the first protocol was replicated but with the addition of 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX) (0.1 µM; antagonist of adenosine receptors). The last experiment compared the ability of 2-chloro-N(6)-cyclopentyladenosine (CCPA) (10 µM; selective A1 agonist), 5'-N-ethylcarboxamide adenosine (NECA) (10 µM; agonist for all adenosine receptor subtypes) and CGS21680 (10 µM; selective A2A agonist) to inhibit LPS-induced TNF-α and CXCL10 production. RESULTS: (1) 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine similarly inhibited LPS-induced TNF-α and CXCL10 production; (2) DPSPX nearly eliminated the inhibitory effects of 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine on LPS-induced TNF-α and CXCL10 production; (3) CCPA did not affect LPS-induced TNF-α and CXCL10; (4) NECA and CGS21680 similarly inhibited LPS-induced TNF-α and CXCL10 production. CONCLUSIONS: 2',3'-cAMP and its metabolites (3'-AMP, 2'-AMP and adenosine) inhibit LPS-induced TNF-α and CXCL10 production via A2A-receptor activation. Adenosine and its precursors, via A2A receptors, likely suppress TNF-α and CXCL10 production by activated microglia in brain diseases.

Seasonal patterns of carbohydrate storage in four tropical tree species.

We examined the seasonal variation in total non-structural carbohydrate (TNC) concentrations in branch, trunk, and root tissues of Anacardium excelsum, Luehea seemannii, Cecropia longipes, and Urera caracasana growing in a seasonally dry forest in Panama. Our main goals were: (1) to determine the main sites of carbohydrate storage, and (2) to determine if seasonal patterns of carbohydrate storage are related to seasonal asynchronies in carbon supply and demand. We expected asynchronies to be related to seasonal variation in water and light availability and to foliar and reproductive phenology. Cecropia and Urera are fully drought-deciduous and so we expected them to exhibit the most dramatic seasonal variation in TNC concentrations. We predicted that maximum carbon supply would occur when canopies were at their fullest and that maximum carbon demand would occur when leaves, flowers, and fruits were produced. The concentration of total non-structural carbohydrates was assessed monthly in wood tissue of roots and in wood and bark tissue of terminal branches. Trunk tissue was sampled bimonthly. All tissues sampled served as storage sites for carbohydrates. As predicted, TNC concentrations varied most dramatically in branches of Cecropia and Urera: a 4-fold difference was observed between dry season maxima and wet season minima in branch wood tissue. Peak concentrations exceeded 25% in Urera and 30% in Cecropia. Less dramatic but significant seasonal variation was observed in Anacardium and Luehea. In all species, minimum branch TNC concentrations were measured during canopy rebuilding. In Anacardium, maximum branch TNC concentrations occurred when canopies were at their fullest. In Cecropia, Urera, and Luehea, TNC concentrations continued to increase even as canopies thinned in the early dry season. The greater photosynthetic capacity of leaves produced at the beginning of the dry season and the potential for the export of carbohydrates from senescing leaves may explain this pattern. In all species, the phenology of carbon gain was more important than the phenology of reproduction in influencing seasonal carbohydrate patterns. The combination of high TNC concentrations and the large biomass of branches, trunks, and roots indicates these species are storing and moving large quantities of carbohydrates.

Photosynthetic responses of Miconia species to canopy openings in a lowland tropical rainforest.

We examined the photosynthetic acclimation of three tropical species of Miconia to canopy openings in a Costa Rican rainforest. The response of photosynthesis to canopy opening was very similar in Miconia affinis, M. gracilis, and M. nervosa, despite differences in growth form (trees and shrubs) and local distributions of plants (understory and gap). Four months after the canopy was opened by a treefall, photosynthetic capacity in all three species had approximately doubled from closed canopy levels. There were no obvious signs of high light damage after treefall but acclimation to the gap environment was not immediate. Two weeks after treefall, Amax, stomatal conductance, apprarent quantum efficiency, and dark respiration rates had not changed significantly from understory values. The production of new leaves appears to be an important component of light acclimation in these species. The only variables to differ significantly among species were stomatal conductance at Amax and the light level at which assimilation was saturated. M. affinis had a higher stomatal conductance which may reduce its water use efficiency in gap environments. Photosynthesis in the more shade-tolerant M. gracilis saturated at lower light levels than in the other two species. Individual plant light environments were assessed after treefall with canopy photography but they explained only a small fraction of plant variation in most measures of photosynthesis and growth. In conclusion, we speculate that species differences in local distribution and in light requirements for reproduction may be more strongly related to species differences in carbon allocation than in carbon assimilation.

Allocation to reproduction in the chaparral shrub, Diplacus aurantiacus.

The semi-drought-deciduous shrub, Diplacus aurantiacus, allocates a large, relatively constant proportion of carbon and nitrogen to sexual reproduction. Experimental manipulation at a site in the chaparral of coastal central California showed that both reproduction and vegetative growth were strongly limited by water and little affected by shade or by addition of nutrients unless accompanied by water. Potential competition for carbon between growth and reproduction is reduced by photosynthesis within reproductive structures; competition is also constrained by localization of translocation. Results are discussed in relation to the hypothesis that allocation to reproduction in Diplacus has been selected to maximize reproductive success.

P7C3 neuroprotective chemicals block axonal degeneration and preserve function after traumatic brain injury.

The P7C3 class of neuroprotective aminopropyl carbazoles has been shown to block neuronal cell death in models of neurodegeneration. We now show that P7C3 molecules additionally preserve axonal integrity after injury, before neuronal cell death occurs, in a rodent model of blast-mediated traumatic brain injury (TBI). This protective quality may be linked to the ability of P7C3 molecules to activate nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in nicotinamide adenine dinucleotide salvage. Initiation of daily treatment with our recently reported lead agent, P7C3-S243, 1 day after blast-mediated TBI blocks axonal degeneration and preserves normal synaptic activity, learning and memory, and motor coordination in mice. We additionally report persistent neurologic deficits and acquisition of an anxiety-like phenotype in untreated animals 8 months after blast exposure. Optimized variants of P7C3 thus offer hope for identifying neuroprotective agents for conditions involving axonal damage, neuronal cell death, or both, such as occurs in TBI.

Detection of Escherichia coli, Salmonella spp., Shigella spp., Yersinia enterocolitica, Vibrio cholerae, and Campylobacter spp. enteropathogens by 3-reaction multiplex polymerase chain reaction.

The magnitude of bacterial diarrhea in developing countries is largely unknown because affordable detection methods are not available. We have developed a polymerase chain reaction (PCR)-based assay for use in areas with limited resources to screen for diarrheogenic strains from clinical isolates. To simplify the assay and minimize reagents, our method implemented the use of plasmids rather than bacteria as template controls and the use of bacterial suspensions or crude DNA preparations rather than purified genomic DNA as template DNA. The assay consisted of 3 PCR reactions using 3 groups of 5 to 6 primer pairs to identify the 11 most common bacterial diarrheogenic pathogens. The 3-reaction multiplex PCR amplifies DNA targets specific for each 1 of the 6 Escherichia coli diarrheogenic strains and the 5 non-E. coli diarrheogenic strains, including Salmonella spp., Shigella spp., Campylobacter spp., Yersinia enterocolitica, and Vibrio cholerae. The assay may provide an important epidemiologic tool to investigate the role of diarrheogenic bacterial pathogens in areas of the world with limited resources.

Life history, enamel formation, and linear enamel hypoplasia in the Ceboidea.

Linear enamel hypoplasia (LEH), a developmental defect of enamel, increases in frequency from prosimian to monkey to lesser ape to great ape grades (Guatelli-Steinberg 2000 Am. J. Phys. Anthropol. 112:395-410, [2001] Evol. Anthropol. 10:138-151; Newell 1998 Ph.D. dissertation, Temple University). This taxonomic pattern in the distribution of LEH is closely related to maturation length across the primate order (Newell 1998 Ph.D. dissertation, Temple University, 2000 Am. J. Phys. Anthropol. [Suppl.] 30:236). Longer maturation periods are associated with higher LEH frequencies; they appear to provide greater opportunity for defects to form. The present study explores the relationship between maturation length and LEH frequency within the Ceboidea. Because of its prolonged period of growth, Cebus is predicted to manifest LEH at a higher frequency than the more rapidly maturing ceboid genera. To test this hypothesis, two separate researchers (E.A.N. and D.G.-S.) examined LEH in nonoverlapping museum series of ceboids. The results support the hypothesis: in 13 genera (n = 1,276), E.A.N. found that LEH frequencies ranged from 0% in Callicebus, Cebuella, and Saimiri to 20% in Cebus. D.G.-S. found similar frequencies among five genera (n = 107), from 0% in Saimiri to 32% in Cebus. Thus, the broad pattern of LEH distribution evident across major taxonomic groups of primates is repeated within the Ceboidea. We also examined a related hypothesis linking the spacing of perikymata, which is influenced by enamel extension rates (Shellis 1998 J. Hum. Evol. 35:387-400), to LEH. The most likely areas of tooth crowns to exhibit LEH in human teeth are those in which perikymata are most closely spaced (Hillson and Bond 1997 Am. J. Phys. Anthropol. 104:89-103). We hypothesized that the longer-maturing Cebus, with its elevated LEH frequency, will also exhibit more closely spaced perikymata than other ceboids. Analysis of a small microscopic subsample (n = 8) lends limited support to this second hypothesis.

Localized hypoplasia of the primary canine in bonobos, orangutans, and gibbons.

This paper extends observations by Lukacs ([1999] Am. J. Phys. Anthropol. 110:351-363; [2001] Am. J. Phys. Anthropol. 116:199-208) of localized hypoplasia of the primary canine (LHPC) among large apes to gibbons, bonobos, and orangutans. LHPC is a roughly circular area of deficient enamel on the labial surface of primary canine teeth which, on current evidence from humans, forms several months after birth due to malnutrition-induced craniofacial osteopenia, leading to crypt fenestration that exposes the dental follicle and more deep-sited ameloblasts to minor physical traumata during normal motor infant development. Our goal was to determine the prevalence of LHPC among a variety of apes which differ in body mass and socioecology, with a view to elucidating the etiology of the defect. We examined juvenile dentitions from 122 animals from three taxa: 8 Hylobates lar, 75 Pongo pygmaeus from Borneo and Sumatra, and 39 Pan paniscus from central Africa. Reported variables include taxon, sex, arcade, side, and tooth size. Presence/absence and ordinal severity of defect expression were recorded by description, microphotography, and scanning electron microscopy. Molds were taken in high-resolution dental impression materials and cast in epoxy resin. There are clear taxonomic, but no sex, differences. Prevalence ranged from 0.0% in gibbons to 61.5% in bonobos and 85.3% in orangutans. The result for orangutans is similar to that reported by Lukacs ([1999] Am. J. Phys. Anthropol. 110:351-363), while bonobos are much more affected than were the common chimpanzees (22%) described by Lukacs ([1999] Am. J. Phys. Anthropol. 110:351-363). There are no significant antimeric differences, but the lower canine is much more affected than the upper by LHPC. We show that larger teeth are more affected by LHPC and have more severe defects. Also, we encountered several instances of patent or healing canine crypt fenestrations, occasionally in direct association with LHPC. Location of the defect indicates that LHPC may occur perinatally but more usually several months postnatally. Histological examination showing the neonatal line and LHPC is required to resolve the issue of timing. We concur with Lukacs ([1999] Am. J. Phys. Anthropol. 110:351-363) that taxonomic, anatomical, and environmental variables combine to determine the occurrence and appearance of LHPC. Nevertheless, we conclude that LHPC probably reflects deficient growth of the arcades in infant apes and humans.