Caenorhabditis elegans intoxication by Crystal (Cry) protein PFTs. We mutagenized and screened for C. elegans mutants resistant to a Cry PFT and recovered one mutant. Complementation, sequencing, transgenic rescue, and RNA interference data demonstrate that this mutant eliminates a gene normally involved in repression of the hypoxia (low oxygen response) pathway. We find that up-regulation of the C. elegans hypoxia pathway via the inactivation of three different genes that normally repress the pathway results in animals resistant to Cry PFTs. Conversely, mutation in the central activator of the hypoxia response, HIF-1, suppresses this resistance and can result in animals defective in PFT defenses. These results extend to a PFT that attacks mammals since up-regulation of the hypoxia pathway confers resistance to Vibrio cholerae cytolysin (VCC), whereas down-regulation confers hypersusceptibility. The hypoxia PFT defense pathway acts cell autonomously to protect the cells directly under attack and is different from other hypoxia pathway stress responses. Two of the downstream effectors of this pathway include the nuclear receptor nhr-57 and the unfolded protein response. In addition, the hypoxia pathway itself is induced by PFT, and low oxygen is protective against PFT intoxication. These results demonstrate that hypoxia and induction of the hypoxia response protect cells against PFTs, and that the cellular environment can be modulated via the hypoxia pathway to protect against the most prevalent class of weapons used by pathogenic bacteria.Pore-forming toxins (PFTs) are by far the most abundant bacterial protein toxins and are important for the virulence of many important pathogens. As such, cellular responses to PFTs critically modulate host-pathogen interactions. Although many cellular responses to PFTs have been recorded, little is understood about their relevance to pathological or defensive outcomes. To shed light on this important question, we have turned to the only genetic system for studying PFT-host interactions— Bacteria make many different protein toxins to attack our cells and immune system in order to infect. Amongst them, pore-forming toxins (PFTs), which punch holes in the protective plasma membrane that surrounds cells, are by far the most abundant and constitute important virulence factors. Since the integrity of the plasma membrane is fundamental to maintaining the normal intracellular environment, the breaching of the plasma membrane by PFTs results in many and dramatic intracellular responses. However, we know little about the relevance of these responses to cell survival or cell intoxication. Here, using the only genetic system for studying pore-forming toxin effects in a whole animal, we show that the same response that protects cells against low oxygen stress unexpectedly also protects cells against pore-forming toxins. Mutations in the animal that hyper-activate the low oxygen response actually make animals resistant to pore-forming toxin attack, whereas mutations that inactivate the low oxygen response make animals more susceptible. Furthermore, a low oxygen environment itself is protective against pore-forming toxins. These data show a new and powerful connection between low oxygen responses and defense against the single most common mode of bacterial attack. Clostridium septicum, streptolysins by Group A and B Streptococci, α-toxin by Staphylococcus aureus, Vibrio cholerae cytolysin (VCC), α-hemolysin from uropathogenic E. coli, cytolysin from Enterococcus faecalis, and crystal (Cry) proteins from Bacillus thuringiensis (Bt).Pore-forming toxins (PFTs) are by far the most abundant and amongst the most important bacterial protein virulence factors 2+ influx, K+ efflux, increased endocytosis/exocytosis, vacuolization, necrosis, and apoptosis Although they are expressed by many bacterial pathogens and are broadly important as potentiators of infection, the effects of these toxins on host cells have been vastly understudied. There are several reasons for this lack of attention. First, their mechanism of action is deceptively simple. Second, most of the attention has been given to understanding how PFTs can change conformation from secreted, soluble proteins to insoluble proteins embedded in the plasma membrane. Third, because breaching of the plasma membrane is a major insult to a cell, a multitude of cellular responses to PFTs have been reported, including CaBacillus thuringiensis (Bt) Crystal (Cry) PFT – Caenorhabditis elegans toxin-host interaction system C. elegans has become an important genetically tractable organism for studying immune responses to bacterial pathogens C. elegansC. elegans allowed for the first molecular PFT defense pathway identified, p38 mitogen-activated protein kinase (MAPK) pathway C. elegans and was subsequently shown to result in loss of protection against PFTs in mammalian cells as well C. elegans and mammals To address many of these limitations, an excellent genetic system for studying PFT responses in an intact animal has recently emerged: the C. elegans mutants hypersensitive to PFTs C. elegans mutants resistant to PFTs. The reason for this assumption is that no intracellular pathway mutants were known that can make cells resistant to PFTs in general. Here we report on the results of a PFT resistance screen and find, unexpectedly, that resistance can be achieved by mutations that up-regulate the C. elegans low oxygen (hypoxia) response. Elimination of HIF-1 (hypoxia inducible factor 1), the main effector of the hypoxia pathway, abrogates this resistance and can lead to PFT hypersensitivity. This protection applies to multiple different PFTs and is clearly distinguished from the role of the HIF-1 pathway in other stress responses and aging. Furthermore, the hypoxia pathway is activated in response to PFTs, and low oxygen is itself protective against PFT attack. Our results indicate that the hypoxia/low oxygen response is likely to be of general importance for cellular responses to small-pore PFTs.Since, when studying intracellular PFT response pathways in the past, we have screened for To identify pathways important for cellular responses to PFTs, we screened for mutants resistant to the PFT Cry protein, Cry21A. Cry21A is a three-domain Cry protein that targets nematodes and is in the same family as Cry5B C. elegans hermaphrodites were mutagenized with EMS and allowed to self-fertilize for two generations. Sixty eight thousand F2 mutagenized hermaphrodites were fed an intoxicating dose Cry21A PFT and then screened for those that resisted intoxication. One mutant line, allele ye49, was identified that bred true and is resistant to Cry21A PFT produced either from Bt or E. coli  and found that ye49/egl-9(sa307) animals are resistant to Cry21A PFT, indicating ye49 fails to complement egl-9(sa307) and most probably mutates the same gene  that upon translation is predicted to truncate the protein in the prolyl hydroxylase domain, thereby eliminating protein hydroxylase function  results in animals resistant to Cry21A  pathway (C. elegans) is hydroxylated by a prolyl hydroxylase  that then increases HIF-1's affinity for von Hippel-Lindau tumor suppressor protein , part of an E3 ubiquitin ligase complex. Association of HIF-1 with VHL-1 eventually leads to HIF proteasomal degradation. When EGL-9 is disabled by mutation, HIF-1 is stabilized at constitutively high levels even under normoxic  conditions The EGL-9 protein is a prolyl hydroxylase and functions in the  pathway . The abiSince loss of EGL-9 function confers resistance to Cry21A PFT, we hypothesized that other elements of the hypoxia pathway might be important as well. We therefore performed quantitative dose-dependent mortality assays using null or putative null alleles of all the above elements of the hypoxia pathway. L4 staged animals from each genotype and wild-type N2 were placed in numerous doses of Cry21A PFT or Cry5B PFT and scored for viability after a few days .egl-9(sa307) and egl-9(ye49) animals are resistant to PFT-induced mortality relative to wild-type animals, with resistance strongest at higher PFT doses , loss of EGL-9 results in 3–5 fold resistance over wild-type animals to Cry21A or Cry5B PFTs  mutant animals are resistant over a similarly wide range of Cry21A and Cry5B PFT doses  mutant animals on Cry21A PFT. RHY-1 (regulator of hypoxia-inducible factor) antagonizes HIF-1 function by inhibiting expression of some HIF-1 target genes via a VHL-1 independent pathway 50 values, animals lacking RHY-1 are 5.7× resistant to Cry21A PFT  and egl-9(sa307) hif-1(ia04) double mutant animals to Cry21A and Cry5B PFTs. When fed Cry21A, hif-1(ia04) animals have a response that is indistinguishable from wild-type animals (egl-9(sa307) hif-1(ia04) double mutant animals have a statistically identical response to Cry21A as wild-type animals at all doses except at 8 µg/mL  and egl-9(sa307) hif-1(ia04) animals on Cry21A PFT are statistically identical (P>0.05) but both statistically different than that of egl-9(sa307)  mutant animals subject to egl-9 RNAi are not (egl-9(sa307) mutants are resistant to Cry21A, this resistance is suppressed by RNAi of hif-1. Thus, HIF-1 is required for Cry21A resistance mediated by loss of EGL-9.To confirm that the resistance associated with  animals ; Table 1 are not . Similari.e., loss of HIF-1 suppresses Cry5B PFT resistance associated with loss of EGL-9). There is, however, one striking difference between the hif-1 results with Cry21A and Cry5B. Both hif-1(ia04) and egl-9(sa307) hif-1(ia04) animals are hypersensitive to Cry5B PFT relative to wild-type animals. That is, animals lacking HIF-1 are more readily killed by Cry5B PFT than wild-type animals, especially at doses ∼5–10 µg/mL  The results with Cry5B PFT are similar to those of Cry21A ; Table 1 P<0.05; ; Table 1C. elegans two V. cholerae strains that differ primarily in their ability to produce another small-pore PFT, VCC. VCC is a virulence factor of V. cholerae and mutants lacking VCC are attenuated for pathogenesis in vivo, especially for strains lacking cholera toxin C. elegans lacks sialic acid that the B subunit binds to as part of its GM1 receptor C. elegans, CVD109(VCC+) is more virulent than CVD110(VCC−), demonstrating that VCC is a virulence factor for C. elegans  animals are resistant relative to wild-type animals (egl-9(sa307) animals are not resistant (hif-1(ia04) and egl-9(sa307) hif-1(ia04) animals are, as with Cry5B PFT, hypersensitive relative to wild-type animals on CVD109(VCC+)  mutant animals are hypersensitive compared to wild-type animals to CVD110(VCC−) strain  protects the animals against VCC and PFTs (hence egl-9 mutants are resistant to the VCC+ strain), activation of the hypoxia pathway may make the animals more susceptible to other V. cholerae virulence factors. The relative contribution to these responses (protection versus susceptibility) is dependent upon which virulence factors are present and their relative contribution to virulence. In the VCC+ strain, the PFT has important function. Hence, the protective role of pathway activation can be discerned. In the VCC− strain, the PFT defense is no longer needed. Hence, the susceptible role can be discerned. It is this give-and-take interaction between the host and virulence factors that could partly explain why constitutive mutation in egl-9 is not selected in the wild. In any event, taken together, our Cry PFT and VCC data demonstrate that stabilization of HIF-1 results in resistance to VCC PFT whereas loss of HIF-1 results in hypersensitivity to VCC PFT.Our results with hypoxia pathway mutants on CVD109(VCC+) and CVD110(VCC−) are striking and parallel those with Cry PFTs. When feeding on CVD109(VCC+), esistant ; Table 2egl-9 mutant animals might show resistance to other stressors as well. We found that, relative to wild-type animals, animals lacking EGL-9 are resistant to killing by 1) the pathogen Pseudomonas aeruginosa PA14; 2) heat stress; and 3) oxidative stress (daf-2 mutant egl-9(ye49) and egl-9(sa307) mutant animals live longer than N2 wild-type when feeding on the standard E. coli strain (Because loss of EGL-9 results in resistance to PFTs (here) and cyanide e stress . Since ci strain , Table 2hif-1(ia04) loss-of-function mutant animals as well as egl-9(sa307) hif-1(ia04) mutant animals are resistant to P. aeruginosa PA14 infection, heat stress, and oxidative stress  animals , is sufficient to rescue the egl-9(sa307) Cry21A resistance phenotype. Control animals in which green-fluorescent protein (GFP) was expressed from the cpr-1 promoter did not result in rescue. Quantitative mortality assays using two independent lines of cpr-1::egl-9-transformed egl-9(sa307) mutant animals confirm that intestinal-specific expression of EGL-9 rescues Cry21A PFT resistance to a level statistically indistinguishable from N2 wild-type (not shown). These data are consistent with the hypoxia pathway acting to directly counteract the effects of PFTs and not, for example, providing protection via altered behavior.Bt Cry PFTs attack intestinal cells  animals , but notC. elegans and asked if any of these are involved in PFT defenses. One pathway immediately surfaced, the unfolded protein response or UPR C. elegansTo address how the hypoxia pathway might function in protection against PFTs, we sought in two ways to find functional downstream effectors of the pathway. First, we compared known functional targets of the hypoxia pathway in xbp-1 mRNA, which is spliced upon activation of the pathway xbp-1 levels in egl-9 mutant animals . However, to date no functional role of nhr-57 for any HIF-1-regulated pathway has been shown.We conversely asked if any of the genes known to be involved in PFT INCED are known to be important for the hypoxia pathway. From over 100 PFT INCED genes we have identified in our lab, we found one and only one currently known to be regulated by the hypoxia pathway, nhr-57 results in animals slightly but statistically hypersensitive to Cry5B PFT . We find that 4 and 8 hours of treatment with PFT significantly induces nhr-57 expression 5.3 and 3.6 fold respectively  oxygen levels minus or plus the presence of E. coli-expressing Cry5B PFT. We find that low oxygen is indeed protective against PFT intoxication in that animals exposed to PFT in a low oxygen environment for 24 hours are significantly healthier than animals exposed to PFT in normoxia (hif-1(ia04) mutant animals exposed to Cry5B PFT do not get any protection when placed in a hypoxic environment  could be achieved by up-regulation of HIF-1 and/or by hypoxia. The identification of the hypoxia pathway as an important PFT INCED pathway thus unexpectedly provides a novel and potentially powerful means of protecting against the single most common mode by which bacterial pathogens attack us.To our knowledge, these results are the first to demonstrate that an intracellular pathway can be altered to promote general egl-9(sa307), hif-1(ia04), egl-9(sa307) hif-1(ia04), vhl-1(ok161), rhy-1(ok1402), the Hawaiian strain CB4856 and HT1593 [unc-119 (ed3)] were obtained from the Caenorhabditis Genetic Center (CGC). All strains were either previously outcrossed or outcrossed here at least six times , rhy-1(ok1402)). egl-9(sa307) is a null allele of egl-9 that carries an internal 243-bp deletion removing part of exons 5 and 6 hif-1(ia04) allele removes exons 2, 3 and 4 of hif-1, including the DNA binding domain, and is believed to be a null allele vhl-1(ok161) allele removes exons 1 and 2 of vhl-1 and is believed to be a null allele rhy-1(ok1402) allele deletes exons 2, 3 and 4 of rhy-1 and is also believed to be null Strains were maintained at 20°C under standard conditions cry21A gene was cloned under 700 bp of the cry6A promoter region and subcloned into the Bt/E. coli shuttle vector pHT3101. The plasmid was transformed into a nontoxic host Bt strain (4D22). Cry21A SCLs were prepared using standard procedures For production in Bt, the Mutagenesis and selection of Cry21A resistance mutants was carried out as described for Cry5B egl-9(sa307) males and dpy-17(e164);ye49 hermaphrodites. As a control, cross-progeny from egl-9(sa307) males into dpy-17(e164) and from N2 males into dpy-17(e164);ye49 were also tested. ye49 was mapped between dpy-11(e224) and unc-76(e911) using standard three-factor mapping. A dpy-11(e224) ye49 unc-76(e911) triple mutant was then made in order to perform single nucleotide polymorphism mapping with the Hawaiian strain (CB4856) Complementation tests were performed by testing F1 progeny from the cross between egl-9(ye49) animals were used to sequence the egl-9 gene. For transformation rescue, a 13.4kb-PCR fragment covering from 3kb upstream to 2kb downstream of egl-9 transcript was amplified with primers GAGCAACTCGTGGGTTTGTT and CTTCCAGAGGCGTTGTCTTC using the LongAmp Taq (Biolabs) from N2 genomic DNA and injected in egl-9(ye49) worms as described Genomic DNA and cDNA prepared from egl-9 rescuing plasmids were constructed by PCR amplification of unc-31 and cpr-1 promoters and then fused to egl-9 and gfp open reading frames using the Multisite Gateway cloning system (Invitrogen) and pCG150 (containing unc-119 rescuing fragment) unc-119(ed3);egl-9(sa307)] by ballistic bombardment For tissue-specific rescue, E. coli toxin assays, we used E. coli JM103 with pQE9 empty vector or a cry21A gene insert under control of the lacZ promoter E. coliFor Cry21A Dose-dependent mortality assays with purified Cry5B were performed as described V. cholerae lifespan assay was performed as described Δ(ctxAB zot ace) and CVD110 rΔ(ctxAB zot ace) hlyA::(ctxB mer) Hg strains, derived from V. cholerae El Tor E7946, were used P. aeruginosa lifespan assays were performed on slow-killing plates as described E. coli strain HT115 expressing double-stranded (ds) RNA  testing on Cry5B PFT was performed slightly differently . Briefly, rrf-3(pk1426) animals were fed E. coli-expressing dsRNA in liquid media with 1mM IPTG at 25°C for ∼30 h. 20 µg/ml of Cry5B or 20 mM HEPES control were then added, as well as 200 µM FUdR. Hermaphrodites viability was scored after 6 days at 25°C.  and 50 µg/mL ampicillin spread with hif-1(ia04) mutant animals were pipetted onto toxin plates spread with 30 µl of a mixture of E. coli OP50 strains expressing or not Cry5B at the ratio 1∶1. Plates were placed immediately in a 2% O2 chamber for 24 hours, while control plates were placed in room air. Images were taken with an Olympus BX60 microscope as described To test worms under hypoxia, L4 wild type and xbp-1 mRNA, we used primers xbp-1_sqf2 GCATGCATCTACCAGAACGTC and xbp-1_sqr2 GTTCCCACTGCTGATTCAAAG to amplify cDNA from wild-type and egl-9(sa307) animals. The forward primer xbp-1_sqf2 anneals to exon 1 and the reverse primer xbp-1_sqr2 anneals to the exon1-exon 2 junction sequence produced when intron 1 is spliced out. The experiment was carried out using two independent sets of cDNA and two repeats within each set. Primers TTATCGAGTTTCTCGCATTGG and AAGTCTGCAATCACGCTCTGT were used to quantify expression of nhr-57. Induction of expression of nhr-57 by Cry5B was tested in glp-4(bn2) animals treated for 1, 2, 4 and 8 hours on E. coli OP50 strains expressing Cry5B or not. The experiment was carried out using three independent sets of cDNA. Normalization in all cases was to eft-2 transcript levels.Real time PCR was performed as described 50 values were determined by PROBIT analysis LCFigure S1egl-9 mutant animals resist Cry21A PFT-induced sterility. Numbers given are the relative brood sizes of wild-type N2 and egl-9(ye49) animals on Cry21A normalized to no-toxin controls (mean of three independent assays). For brood size assays, L4 hermaphrodites from N2 and egl-9(ye49) were picked one each to four to six plates and incubated at 20°C. Every 24 h, the originally picked worms would be picked to a new plate; progeny from the old plate were counted 24 h later. This process was continued until the original parents ceased to produce progeny. On E. coli plates expressing Cry21A, N2 animals show a 6.2-fold reduction in fertility, while egl-9(ye49) show only a 1.5-fold reduction in fertility. Error bar represents standard error of the mean. P = 0.03 (one-tailed T test).(0.87 MB PDF)Click here for additional data file.Figure S2rhy-1 mutation confers resistance to Cry21A PFT. Dose-dependent mortality assays were performed using Cry21A spore crystal lysates to quantitatively compare sensitivities of wild-type N2 and rhy-1(ok1402) mutants. Each data point shows the mean and standard error of the mean from three independent experiments . Statistical differences between mutant strains and N2 are given for each concentration using P values represented by asterisks as follows: * P<0.05; ** P<0.01; *** P<0.001. LC50 values and % alive at specific doses are reported in (1.98 MB PDF)Click here for additional data file.Figure S3egl-9(sa307), and hif-1(ia04) mutant animals were treated with RNAi of either empty vector (L4440), egl-9, hif-1 or dpy-3 (positive control for RNAi effectiveness) and exposed to E. coli expressed Cry21A PFT for 48 hours. When put on toxin plates, only wild-type animals on egl-9(RNAi) and egl-9(sa307) on either empty vector, egl-9(RNAi), or dpy-3(RNAi) display a resistance phenotype. RNAi of hif-1 in the presence of egl-9(sa307) suppresses Cry21A PFT resistance. RNAi of egl-9 in the presence of hif-1(ia04) does not confer resistance to Cry21A. Scale bar is 0.5 mm.RNAi confirmation that Cry21A resistance associated with loss of EGL-9 is mediated through HIF-1. Wild-type N2, (4.07 MB PDF)Click here for additional data file.Figure S42) for 72 hours (bottom two rows). Worms co-treated with hypoxia and Cry5B are significantly healthier  than worms treated with Cry5B under normoxia. Scale bar is 0.2 mm.Hypoxia confers protection against Cry5B PFT. Resistance to Cry5B PFT was compared among wild-type N2 worms in normoxia (top two rows) and in hypoxia (1.5% O(0.55 MB PDF)Click here for additional data file.Table S1Data and analyses of VCC, PA14, and lifespan repeat assays.(0.10 MB PDF)Click here for additional data file.


A series of experiments on rats have been performed, to study the effects of long time (50 days) exposure to electromagnetic fields of extremely low frequency  and amplitude , testing whether the metabolic processes would be affected. The background lies on recent observations on the behaviour of isolated enzymes in vitro exposed to EFL fields. In these experiments, the cyclotron (or Larmor) frequency of the metallic ion has been used to "stimulate" the metalloproteins redox-active site, thus obtaining a clear variation of the enzyme functionality. In this paper we have extended for the first time the check to more complex animal metabolism. The novelty of this approach implies that a large amount of data had to be analyzed since it was not possible, in principle, to select only a few parameters among all the potential effects. Several biochemical parameters have been evaluated by comparing their values during the periods of exposure (field ON) and non exposure (field OFF). The evidence that long term exposure to electromagnetic fields with a well defined frequency may have relevant effects on parameters such as body weight, blood glucose and fatty acid metabolism has been obtained. Exposure to artificially generated electromagnetic (e.m.) fields is a common occurrence for a large number of individuals: its biological consequences, still largely unknown, are being studied in experimental animals -4 and inc q and m are respectively the electrical charge and the mass of the ion, and B0 is a constant magnetic field. Furthermore, according to Zhadin and co-workers [ac, which has a frequency matching the cyclotron frequency fc. Such a mechanism should be a good candidate to explaining the selective enhancement of the ions flow through the cell membranes, and would consequently affect the rate of biochemical reactions and the enzymatic activities.In the '80s, it has been recognized ,10 that -workers ,12 an iom and charge e in a constant magnetic field of strength B0 leads to the superposition of a uniform precession of angular frequency fL (known as Larmor precession) about the direction of the field on the original motion. The Larmor precession provides a mechanism by which biological systems become sensitive to small static and resonating magnetic fields extensively described by Edmonds [More recently, these observations have been checked by applying e.m. fields to isolated enzymes studied in vitro. The cyclotron (or Larmor) frequency of the metallic ion has been used to "stimulate" the metalloproteins redox-active site . The sim Edmonds . As cleaIn order to investigate the possible consequences of such findings on living organisms, a series of experiments with rats exposed to fields of very low intensity and frequency were carried out, aimed at testing whether such non-thermal e.m. fields may influence the basic metabolic processes. The novelty of our approach leads to the analysis of a large amount of data since it was not possible, in principle, to select only a few parameters among all the potential targets. In these experiments, it has been observed that biochemical and morphological parameters have been affected by 50 days exposure, even though the amplitude and frequency of the field was largely under the threshold usually considered to be effective by the medical community. In order to maximise the likelihood that preliminary experiments might reveal biological effects of some relevance, aging animals were also studied, considering that adult animals may be more resistant to external interventions than aging animals.Experiments on the effects of the ELF (Extremely Low Frequency) weak electromagnetic fields have been performed by using the electronic equipment QUEC-PHISIS QPS1™, provided by PROMETEO S.r.l. .The electronic device being used is able to generate waves in the range where the cyclotron frequencies of the ions involved in metabolic processes lie. Moreover, it is well known from clinical impedencymetry  that e.mThe magnetic field produced inside the cage was measured during the exposure with a gauss meter F.W Bell (Sypris Q3 Test and Measurements) Mod. 7010 equipped with a low field Hall sensor MOX 71-2506-05, with the resolution of 0.1 nT.μT, in order to stimulate the Ca2+ dynamics. The amplitude was set equal to the amplitude of B0 in order to accomplish to the range of validity of the Edmonds model.In order to study the influence of a prolonged exposure to these fields, initially one coil of 75 cm of diameter was used, which allowed the exposure of two rats in one cage positioned just in the middle of the coil Such a dimension let us to dwell easily a cage for long time, thus avoiding disturbing the animal's daily life. The field produced had a frequency of f = 31.6 Hz and an amplitude of about 40 2+ ion dynamics. In fact the q/m ratio of Ca2+ ion is 4.81 × 10-6 Coul/kg, the corresponding fc/B0 is 0.76 (Hz/μT) and the resonant frequency has been calculated being the measured geomagnetic field of 41.5 μT.In a subsequent experiment, a set of identical Helmoltz coils arranged on a rack Fig.  was asse0 magnetic field amplitude, accurately measured during the exposure, ranged between 35.5 and 51.6 μT, as it can be seen in details in Fig. 3+ and Mn3+ having an fc/B0 ratio quite similar to the Ca2+ might have been activated.However, the peculiar geometry and the materials of the rack and of the grids of the cages (stainless steel) did not allow a uniform static magnetic field level to be reached neither along the ax of the rack nor on the plane of the five cages. Thus, the BThis Research Project was communicated on July the 7th 2005. to the Ministry of Public Health as stated by Laws (D.lgs 116/92).ad libitum with water at disposal, was utilised.The animals were kept in stabulary controlled conditions all over the duration of the experiments : temperature 21°C ± 1; hygrometry between 40% and 70%; air change per hour 8 to 12 volumes; nyctohemeral cycle 12 h/12 h; lightning 300 to 600 lux at 1 meter over the floor; noise < 60 db. The cages bases were made out of polypropylene while the ceilings were in stainless steel. In each cage up to 4 adult animals, or 3 aging animals, could be housed. A complete maintenance diet for rats , administered dry In a preliminary experiment two WISTAR SPF(Specific Pathogen Free)/VAF(Virus Pathogen Free) male rats (age at the beginning 50 weeks) were exposed to a field specifically designed to modify calcium dependent enzymatic activities as indicated in the previous paragraph, for 35 days continuously. The animals were then observed without any other intervention for 182 days after the exposure. The limited number of animals used was due to the necessity of a constant exposure and the availability of a small set up. Furthermore, two animals of the same strain, used as controls, were kept in the same conditions without exposure.In the main protocol, 13 male OFA (Oncins France Strain) SPF/VAF rats, from Charles River Laboratories Italia , were exposed for a total time of 50 days.st blood drawing . After this first drawing 2 adult rats and 1 aging rat died. From 2nd to 29th day, all rats were subjected to continuous irradiation by low intensity and frequency electromagnetic fields (ELF) (field ON); on 15th and 29th day respectively, 2nd and 3rd blood drawing was carried out. From 30th to 36th day ELF were deactivated (field OFF); on 36th day 4th blood drawing was carried out. From 37th to 50th day of trial ELF were reactivated (field ON); on 50th day 5th blood drawing was carried out. After the third and fourth drawing two other aging rat were lost. On 50th day of trial all rats were sacrificed by intracardiac inoculation of embutramide + mebezonium iodide + tetracaine hydrochloride.Eight animals were 8–12 weeks old, and Eight animals were 68–72 weeks old. They were hosted into the cages according to the scheme: 4 adult rats in cage 1; 4 adult rats in cage 2; 2 aging rat in cage 3; 3 aging rats in cage 4; 3 aging rats in cage 5. After 15 days of adaptation, all rats were subjected to 1After each blood drawing the rats returned to the same cage. During the experiment, it was decided to submit to euthanasia subject 3 of cage 5 before the others for its health status, while rat 2 of cage 4 died twenty days before the end of the trial.st, 15th, 29th, 36th, and 50th day together with blood drawing, and the amount of food consumed by each group was noted during the trial.The individual body weight was controlled on 1The data shown in the Figures are the averages on the animals which survived along the whole duration of the experiment, i.e. 6 adult and 5 aging rats.μm thickness, and stained with H&E. Liver sections were also stained with periodic acid-Schiff (PAS)/diastase techniques to assess hepatic glycogen.A complete post-mortem examination of all the animals was performed after euthanasia and lungs, heart, spleen, liver, pancreas, kidneys, skeletal muscle and brain samples were fixed in buffered 10% formalin. Half of the brain, ocular tract and eyes were frozen and stored at -80°. Tissues were then processed in routine manner for histological evaluation, cut at 5 2-EDTA were utilized to determine several haematology and biochemical parameters (see below). Blood sampling was performed in animals with no restriction to food availability. In order to reduce variability due to recent food uptake, care was taken to obtain samples at the same time of the day, namely between 3 and 4 pm, i.e. 7–8 hours after light start.All blood samples were obtained by intracardiac drawing after anaesthesia with tyletamine hydrochloride and zolazepan hydrochloride combined with xilazine hydrochloride. Blood samples added to K2-EDTA, by ADVIA 120 HEMATOLOGY SYSTEM  provided with specific veterinary software for rats.Haematological parameters  were measured in blood samples, added up with K2-EDTA, by automatic analyser ROCHE HITACHI 912 PLUS .Biochemical parameters (Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Creatine kinase (CK), creatinine, cholesterol, glucose, total protein, tryglicerides, urea) were measured in plasma samples, obtained after centrifugation of blood samples added with Kω9), linoleic acid (C18:2ω6), linolenic acid (C18:3ω3), eicosatrienoic acid (C20:3ω9), arachidonic acid (C20: 4ω6), docosaesanoic acid (C22: 6ω3), and nervonic acid (C24: 1ω9)), derived as methyl esters, were measured in plasma samples according to Carnielli [Fatty acids , stearic acid (C18:0), oleic acid  and a relatively long follow up (182 days), both animals reached a weight of over 900 grams, while the two control animals reached a value of about 400 grams.2+, Mn3+ and Co3+ might be involved. Such a circumstance makes it more difficult to relate the effect observed on metabolism to a specific ion and, trough the ion to a specific metabolic reaction. Furthermore, exposure was applied for selected periods (ON) followed by a short periods without any exposure (OFF). Everything but the AC field was kept constant during ON and OFF periods. A control group could not be used in this experiment, thus variations between on and off periods were considered as a built-in control. The OFF periods are quite well visible as changes in the rate of the monitored issues: they signal a different adaptation time of the organism in case of an external perturbation. Furthermore, the strain of rats we used in this experiment are very well characterized in their physiological parameters, hence what we observed and reported here can be compared with a large amount of historical data. Among a number of parameters recorded, in this paper are only discussed which who showed striking variations.After this exploratory experiment another experiment was performed by using animals of two different ages. In this case, exposure was not characterized by a unique frequency of exposure field, because of the disposal of the cages along the rack as discussed above, but a quite broad number of ions, including CaWith regard to body weight, the initial values observed were in agreement with the historical set (338 ± 19 gr. for adult and 599 ± 46 gr. for aging rats). Exposure appears to modify severely such an integrated parameter, as shown in Figure Exposure appears to modify also blood glucose content, both in adults and in aging rats, as shown in Figure The effect of e.m. field on lipid metabolism is the most interesting finding of this research. Cholesterol and triglycerides appear to be only marginally affected by the stimulus applied, in fact, both parameters did not change throughout the experiment in both groups, however total free fatty acids and single fatty acids analysis showed visible changes. While exposure did not change substantially these parameters in adult animals, clear differences between the initial values and the final ones appear in aging animals. Figure With reference to haematology data, samples were available only for some of the rats, however in general terms we observed that leukocyte count tends to decrease during the experiment.Finally, it is interesting to mention that at post-mortem examination no relevant gross findings were observed. In fact, most noticed lesions, in both age groups, are minimum blood collections in the pericardium  and in the thorax (5 rats). In 3 rats hemorrhagic findings were seen also in other sites. These findings are related to blood sampling and euthanasia.Main findings of histopathological studies can be summarised as follows. A frequent finding was hepatic glycogenosis: PAS/diastase showed a mild multifocal staining in young animals (12 weeks old), while glycogen amount in hepatocytes cytoplasm was higher and diffusely distributed in 72 weeks old rats. Other changes did not show any significant difference between the two groups.The ultimate goal of our research is the possible application of electromagnetic fields in the treatment of human and animal diseases, where ionic regulation of enzymatic activities have been altered and their restoration to normal is potentially useful for the patient. The present results give several helpful suggestions, both in terms of selection of possible patients and in terms of safety in order to reach this purpose. The present experimental work was carried out in order to have a coarse evaluation of the effects of the exposure, thus no defined experimental end point was selected a priori. Such an approach was mainly due to the fact that this kind of exposure had not been previously studied by other researchers and there is no available literature. This aspect not only makes it difficult to compare our findings with other results, but it also influenced the experimental design, preventing the possibility to choose in advance the most significant parameters to be measured. Nevertheless, the novelty of the observations and their relevance  deserve, in our view, the preparation of the present report.μT and a frequency of about 31 Hz is reported here for the first time. The animals exposed had their weight doubled as compared to the weight of control animals of the same strain, while no other negative effect has been observed in their physiology after 182 days of follow up and during the post mortem examination. This effect has been observed in two different experiments even though the second group was exposed for 50 days with an interval of 15 days (field OFF) and the weigh increase (about 150 grams) was observed only in adults and not in aged animals. The weight increase observed is clearly higher than expected on the basis of the historical data available for these particular strains in the IFFA CREDO database.First, a huge effect in the growth of rats exposed to e.m. field having an amplitude of 40 Second, changes in several metabolic parameters as a consequence of exposure to e.m. fields have been also observed. The frequency of the field was such to "stimulate" several ions in the sense of the cyclotron frequency. These ions may be involved in the enzymatic chains experimentally affected. This issue needs to be analyzed in detail in the future through well designed experiments.Even though the data available are not exhaustive, because of the limited number of animals observed up to now, no major pathological signs related to exposure have been observed in post mortem analysis. Indeed, as far as authoptic findings are concerned, most of the findings described were noticed in both groups, independently from age, and they could be iatrogenic or aging-related. In fact, gross hemorrhagic lesions and some of the described microscopic changes  are possibly related to intra-cardiac blood sampling and to injections of anaesthetic drugs. In particular, changes observed in nervous tissues could be due to an early mild hypoxic condition .From a regulatory point of view, the intensity of the fields applied is considerably below the threshold risk value, our preliminary new data seem to agree with this view and at present there is no reason for concern. From the point of view of patient selection, our findings cannot be directly extrapolated to human pathologies. In general terms, aged animals appear to be more sensitive to the exposure: major changes have been observed in this group and not in the adult animals. In particular the derivatives of the individual responses are much higher in the ON-OFF transient especially for aging animals, In order to explain these observations, we suggest that adult young animals have the capability to reach homeostasis even in the presence of external stimulation, like the one we applied, while aging animals have a reduced capability to cope with external stimulation and thus measurements of several variables are likely to reveal alterations.By analyzing the observed changes in aging animals, it can be said that: a) body weight increased during the first and the second period of exposure and decreased in the off period this results confirms the first experimental run described; b) blood glucose substantially increased in this group more than in young animals; c) the circulating levels of specific free fatty acids clearly show the effect of the exposure.It is interesting to mention that in a recent paper Torres-Duran et al  showed aKnowing that the total free fatty acid content of the blood may be affected substantially by stress, the data were analyzed as the ratio between different fatty acids, in particular as the ratio between single acids and their metabolic precursors. By applying this kind of analysis, the most interesting findings were related to arachidonate/linoleic ratio, most likely a direct consequence of the enzymatic activity of elongase and desaturase. The baseline of aging rats showed substantially higher values than in adults, likely indicating enhanced conversion, furthermore, exposure did not substantially modify the values in adult rats, while decreased the ratio in aging rats, thus bringing the ratio closer to that observed in adults. The importance of such a difference in the comparison between adults and aging rats is further outlined by the fact that other calculated ratios, such as eicosatrienoic/oleic acids ratio and docosaenoic/linolenic acids ratio, were different neither at baseline, nor after exposure.In conclusion, we provide initial evidence that long term exposure to well defined electromagnetic fields may have relevant effects in mammals, thus affecting parameters like body weight, blood glucose and fatty acid metabolism. The present findings have to be confirmed and extended to fully understand their importance; nevertheless, they indicate that this line of research may lead to important results. The clinical consequences of these observations cannot be precisely estimated yet, even tough they point out towards a rational target for possible therapeutic interventions, to be analysed in further studies.Authors disclose any financial and non-financial competing interests  related to the publication of the manuscriptGG carried out the experiments and helped to draft the manuscript. ADN analyzed the results and drafted the manuscript. MP analyzed the results and drafted the manuscript. VF carried out the experiment and helped to draft the manuscript. FB supervised the electronic equipment. SM carried out the histopathological studies. DB analyzed the results and helped to draft the manuscript. GT conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript


The objective of this study was to compare the utility of Thin-Prep (TP) cytologic preparation with that of Cell Block (CB) preparation in the diagnosis of thyroid lesions, mainly follicular epithelial lesions, by fine needle aspiration biopsy (FNAB). Feasibility of using the TP slides for immunocytochemical stains is also discussed.A total of 126 consecutive cases of thyroid FNAB with TP slides and 128 consecutive cases of thyroid FNAB with CB slides were reviewed blindly by two cytopathologists. The presence of colloid, follicular cells, macrophages and lymphocytes/plasma cells were recorded and scored 0–4 on each case based on TP or CB slide review. The cytologic diagnoses were grouped as follows: cyst, colloid nodule, colloid nodule with cystic change, chronic thyroiditis, atypical/neoplastic and non-diagnostic.The TP slides had higher diagnostic rate than CB slides. The diagnostic yield was 68% of the TP slides whereas only 24% of the CB slides were diagnostic. Also, only 4 atypical/neoplastic lesions were diagnosed on the TP slides and the corresponding direct smears, while 5 cases of atypical/neoplastic lesions were diagnosed on the smears but could not be diagnosed on the corresponding CB slides. Additionally, the TP slides revealed cytologic features that were not observed on the direct traditional smears of the same case.In thyroid FNAB cases, TP slide preparation is superior to CB slide preparation and is more likely to have greater cellularity for diagnosis and detect atypical/neoplastic thyroid lesions, particularly those of follicular cell origin. Furthermore, TP slides appear to detect helpful diagnostic cytologic features and should be considered complementary to, rather than replacing, direct smears. FNAB is a cost effective and perhaps the best procedure for the initial evaluation and diagnosis of thyroid lesions ,2. It isA major advantage of the CB method is the ability to perform multiple immunocytochemical stains or other special stains if needed . HoweverWe retrieved the slides and cytology reports of the first 126 cases of thyroid FNA biopsies since converting to the TP method, and also the last 128 cases of thyroid FNA biopsies made with CB method. These FNAB cases were all office procedures done by experienced endocrinologist, with or without ultrasound guidance, usually using 22 or 23 gauge needles connected to a 10 c.c. syringe. The direct smears were mostly alcohol fixed (spray fixation) and stained with Papanicolaou stain. In few cases, half of the smears were air-dried and stained with Diff-Quik stained. The remaining aspirate specimen was rinsed in Saccomono's fluid for CB preparation. After converting to the TP method, the remaining aspirate material was rinsed in the physician's office in buffered fixative, Cytolyt Solution . In the lab, the specimen was spun down, resuspended in a buffer PreservCyt (Cytyc Corporation) and then processed in Thin Prep Processor 2000 (Cytyc Corporation). The TP, CB and direct smear slides were evaluated blindly by two independent cytopathologists without knowledge of the original diagnosis. The examined slides were scored 0–4 on specific cytologic features as follows: cellularity ; colloid ; macrophages ; and lymphocytes/plasma cells . The individual value of each cytologic feature was entered in a designed grid immediately after microscopic examination. The cytologic findings of both pathologists were compared and had high interobserver agreement. In few cases, there were minor differences in scoring, the slides of these cases were jointly reviewed and diagnostic concurrence was obtained. The final cytologic diagnoses for both the TP and CB on each case were classified into the following categories according to previously published cytologic criteria: 1. cyst, 2. colloid nodule, 3. colloid nodule with cystic change, 4. chronic thyroiditis, 5. atypical, 6. neoplastic and 7. nondiagnostic.Table The TP slides were more likely to detect colloid nodules and chronic thyroiditis. There were 52/126 (41%) TP slides diagnostic of colloid nodules/colloid nodules with cystic change compared to only 10/128 (8%) diagnosed on CB slides. Most of the cases had more follicular cell groups on TP slides than on the direct smears of the same case  of TP slides displayed cytologic features consistent with chronic thyroiditis, in contrast to only 4/128 3%) of CB slides  of the cases, while CB slides were diagnostic in only 31/128 (24%) of the cases. The CB and TP slides had similar diagnostic yield only in cases of degenerative thyroid cysts, 17/128 (13%) vs. 16/126 (13%), respectively. In cases of colloid nodules or colloid nodules with cystic change, CB had diagnostic yield of 10/128 (8%) compared to 52/126 (41%) on TP slides. We also found that CB slides were diagnostic of chronic thyroiditis in only 4/128 (3%), while TP slides were diagnostic in 14/126 (11%). More significantly, we found that 5 CB slides failed to identify atypical/neoplastic lesions, including 2 follicular neoplasms that were diagnosed on the corresponding smears. On the other hand, 4 TP slides detected atypical/neoplastic lesions, including 1 papillary carcinoma, that were also diagnosed on the corresponding smears. This sharp contrast is perhaps the most important finding in our study showing that TP slides are more likely to detect atypical/neoplastic lesions than CB slides in thyroid FNAB cytologic evaluation.Our results are essentially in agreement with those of previous studies which demonstrated good diagnostic yield of TP slides of thyroid aspirates. Frost et al found that TP slides of thyroid aspirates have an 85% diagnostic accuracy, and that preparation of only two TP slides is sufficient for accurate cytologic interpretation . Yet, inA major advantage of CB preparation is the ability of performing special stains or immunocytochemical stains if needed . This haSome authors have also elaborated on the cytologic features of specific thyroid lesions, such as follicular lesions and papillary carcinoma, on TP slides of thyroid FNAB specimens and described the differences from the features seen on traditional smears or CB slides ,27.In summary, we conclude that TP slide preparation in thyroid FNAB is more useful than CB slide preparation. Our study shows that the diagnostic yield of TP slides is at least twice that of CB slides in detecting thyroid lesions on FNAB, including atypical/neoplastic lesions, except for degenerative cysts. We further believe that TP slides should be made in addition to but not in lieu of direct (Diff-Quik and Papanicolaou stained) smears as they complement each other.FNAB: fine needle aspiration biopsyTP: Thin PrepCB: cell blockThe authors verify that they have no connection or interests to any company or product mentioned in this article.


Derryck Klarkowski and Daniel Orozco describe the Médecins Sans Frontières program for monitoring the quality of microscopy for malaria, pulmonary tuberculosis, and leishmaniasis. The international humanitarian medical aid organization Médecins Sans Frontières/Doctors Without Borders (MSF) supports a wide network of medical laboratories in resource-constrained countries. Although MSF has always prioritized quality control (QC) for laboratory testing, prior to 2005 we were constrained by two significant limitations. First the QC workload was unsustainable in many programs, as MSF used the traditional protocol of reexamining 10% of negative slides and all positive slides. This is no longer considered practical In May 2005, MSF Operational Centre Amsterdam (MSF-OCA) developed and implemented a standardized, centrally reporting QC program to monitor the quality of microscopy for malaria, pulmonary tuberculosis (TB), and leishmaniasis. The malaria component of this protocol has been adapted by the World Health Organization (WHO) as the recommended international standard for malaria microscopy QC The QC protocol was designed to (1) have a small sample size to be feasible across all settings; (2) enable reliable analysis; (3) monitor both false-positive (FP) and false-negative (FN) results; and (4) be applicable to all microscopy testing.The MSF-OCA protocol is based on a sample size of ten slides/month/site (for each test), as field experience has demonstrated that this QC workload is sustainable in most settings, and on the premise that it is better to perform less QC well than more QC poorly. A small sample size is also important to avoid overloading the limited capacity of the reference laboratory in many resource-constrained settings. Programs are encouraged to include more QC slides if this can be achieved without compromising the quality of the reexamination.In summary, each month for each test: (1) Five weak positive slides are selected randomly from all weak positive slides; or if <5 weak positive slides, then all weak positive slides are selected. (2) Five negative slides are selected randomly from all negative slides; or if <5 negative slides, then all negative slides are selected. (3) If there are <5 weak positive (or negative) slides, then the number of negative (or weak positive) slides is increased to give a total minimum sample size of ten. (4) Strong positive slides are excluded from selection in the QC sample. (5) Laboratories unable to perform QC on a minimum of ten slides are assessed on an individual basis.Blinded QC slides are reexamined within 4 weeks in the field by either a reference laboratory or an independent skilled laboratory technician.Weak positive slides are defined as ≤9 trophozoites/acid-fast bacilli (AFB)/10 high power fields. These definitions were consistent across laboratory sites. Postimplementation experience now suggests that the criteria for a weak positive should be reduced to ≤9 trophozoites/AFB/100 high power fields.While small sample QC has the important advantage of practicality, maintaining reliable analysis is also essential. To compensate for the small number of QC slides reexamined each month, our QC protocol uses analysis of cumulative data over 4-month periods , referred to here as “cohort analysis”. These 4-month cohorts are used to increase the sample size analyzed, and as a compromise between the greater statistical stringency of analyzing a larger number of results over a longer duration  versus the greater immediacy of detecting real-time laboratory performance by analyzing QC over a shorter period.To enable FP analysis on small samples, our protocol uses biased sampling to increase the number of positive slides available for reexamination, and the targeting of weak positive slides to increase discriminatory power.QC protocols that use a small sample size with random sampling of all slides, such as lot quality assurance sampling (LQAS) The protocol selects only weak positive slides because errors of false positivity are most likely to occur during routine microscopy through microscopists reporting negative findings as weakly positive (to be “on the safe side”) However, because FP results are more likely to occur among weak positive slides, reexamining only weak positive slides  may overestimate the FP frequency in routine microscopy. We correct for this by using the formula:A primary objective for MSF-OCA was to develop a protocol that could be used for all microscopy testing. Although LQAS is recommended by WHO and others for AFB direct-smear TB analysis All QC results were reported to the central office in Amsterdam, which enabled comparative monitoring of results across all programs and the identification of poorly performing laboratories. Summarized analysis was reported back to the field to enable individual laboratories to compare their performance to other laboratories in similar settings.We use percentage agreement because it is simple, direct, and understandable at all levels In contrast to stable programs, such as government health laboratory networks, MSF operates as an emergency humanitarian organization, and laboratory programs open and close according to changing priorities. Therefore the QC analysis presented here reflects the overall performance of MSF-OCA programs over 2005–2008 with a changing composition of laboratories.Because only a limited number of laboratories performed leishmaniasis testing, these findings are not presented here.To improve statistical reliability, we only analyzed percent agreement on cohort data that included at least three monthly reports in the 4-month period, and FP and FN on cohort data that included at least ten positive or ten negative slides, respectively . Fifty-sDuring the reported period, the internal MSF-OCA standards were set at ≥95% agreement for all slides (percent agreement) and ≤5% FP and FN slides.Tests of difference between two proportions were performed using the Pearson's Chi-squared test. Analysis was performed using Epi Info 6 (US Centers for Disease Control) and STATA version 8.2 (StataCorp).p<0.001) in the proportion of laboratories meeting each QC target, with the results of 95.7% (45/47), 86.7% (13/15), and 91.3% (42/46), respectively.Marked progressive improvement in the overall malaria microscopy QC performance was seen over the period ; Table 2p = 0.033) and 77.1%  of laboratories, respectively, met these targets. In contrast, the FN frequency remained relatively constant throughout the period (p = 0.527).Progressive improvement in the overall AFB microscopy QC performance was seen over the period ; Table 2e period , with no We found the design of our QC protocol to be practical in field settings and easily understood and implemented by laboratory staff with limited training. We attribute this to a combination of a small QC sample size, a fixed number of slides independent of the workload, and use of simple percentage agreement for statistical analysis. The small sample size considerably decreased the QC workload while maintaining statistical reliability by using targeted sampling of only weakly positive slides and 4-month cohort analysis.Our findings show a significant improvement in the accuracy of malaria and AFB microscopy comparing the periods May–December 2005 and January–August 2008. We attribute this improvement to the strengthening of our protocols, field support, and training over this period. However our QC protocol also played a central role by providing key information on a timely basis allowing us to prioritize those laboratory support activities. Also, and we believe critically, the reporting of compiled data back to the field provided the laboratories with clear performance indicators and enabled field laboratories to directly compare their performance against other laboratories working in similar circumstances. In our experience, this generated an environment of positive “competition” among laboratories that we believe has also contributed significantly to the improvement in laboratory quality performance.For malaria microscopy, the number of FP and FN results decreased markedly. We attribute this to active follow-up of poorly performing laboratories identified by the QC protocol. In contrast, the frequency of FN results for AFB microscopy did not change significantly, and the improvement in percentage agreement reflects the decrease in the frequency of FP results. Laboratories for AFB also entered the analysis period at a higher level of performance compared with malaria microscopy . This may be because AFB microscopy is relatively easier to perform than malaria microscopy as accurate malaria microscopy requires greater microscopy resolution and has a technically more demanding staining procedure.However we also speculate that the random selection of negative AFB smears, which is the standard methodology for AFB QC protocols and is used in our protocol, may be problematic. Saliva smears are in general more likely to be negative or have an AFB density below the threshold of microscopy detection than sputum smears For the future, we are currently incorporating clerical error monitoring into our laboratory QC protocol, as this can also be a major source of error. With the increasing emphasis on disease eradication, we are also developing QC protocols to accommodate low positivity. Finally, we have implemented a pilot study to exclude saliva smears from the AFB QC sample.From this recent field experience, our laboratory QC protocol was found to be well accepted and understood by all levels of field staff, practical in a wide variety of contexts, able to improve performance, and able to provide valuable program management information. As with all QC, implementation and sustainability requires commitment from field staff and project managers. Ongoing supervision and support are critical for central monitoring, ensuring compliance, and regular feedback reporting. The implementation of this centralized-reporting, standardized QC program has provided the catalyst for MSF-OCA to develop a laboratory “culture of quality” over the past 3 years, which in turn has strengthened the commitment and interest of laboratory field staff to ensure the success of health care programs.


Tracheal defects may occur after trauma or prolonged intubation. Resection of tracheal tumors also poses a major challenge for substitution. In an effort to solve this problem, different techniques have been tried with little success. We report on a new animal model which showed acceptable results with fewer complications.We replaced 5 cm of cervical trachea in 10 dogs with harvested infra-renal aorta and repaired the aortic defect with Dacron graft.Necropsy of the grafted aorta and anastomotic site revealed well healed anastomosis in all animals together with ciliated columnar epithelium coverage of grafted aorta and neovascularization of aortic wall.Aortic graft is preferable to other substitutes because of less antigenicity, less vascularity, and no mucous secretions or peristalsis Tracheal defects may occur after trauma or prolonged intubation. Resection of tracheal tumors also poses a major challenge for substitution. In an effort to solve this problem, different techniques have been tried with little success. The various tracheal substitutes and techniques of reconstruction were analyzed by Grillo , who claEleven healthy dogs ranging from 18 kg to 24 kg in weight were studied. All animals received care in compliance with the "Guide for the care and use of laboratory animals" prepared by the Institute of laboratory animal resources, National research Council, and published by the National Academy Press, revised 1996. Five centimeter of cervical trachea was resected and replaced by harvested abdominal aorta. The aortic defect was repaired with Dacron graft.General anesthesia was induced with thiopental and maintained with oxygen and 1–3% halothane through an endotracheal tube. All animals were perfused with a crystalloid solution. Pulse were monitored Introperatively. One gram of Cephazoline was injected after the induction of general anesthesia for wound prophylaxis.A laparatomy was performed thorough a midline abdominal incision to expose at least 5 cm of infrarenal abdominal aorta. Before aortic clamping, 100-unit/kg heparin was given intravenously and 5 cm of aorta was resected circumferentially and replaced by Dacron graft using a running 4-0 Polypropylene suture. The average time for the harvesting and anastomosis was less than 20 minutes without a need for aortic shunt. The excised aortic segment was placed in heparinized blood solution. The abdomen was then closed in two layers by 0-0, 3-0 nylon sutures. Access to the cervical trachea was gained through a longitudinal vertical neck incision by splitting the anterior neck muscles. The trachea was exposed and a 5 cm long window shape defect is made with resection of anterior tracheal rings. The posterior membranous portion of trachea left intact. A longitudinal incision was made along the whole length of harvested aorta [Fig. In post operative day one diet started for the animals and advanced. Clinical examination was performed daily for fist two week post op and then every month including overall status, weight, respiratory status and injury to the laryngeal nerve. Because none of the animals developed respiratory problems or fever and there was not stent, bronchoscopy and chest X-ray was not performed in the post operative period.Animals were sacrificed between two and six months after the procedure with intravenous injection of potassium chloride and Propofol to remove the grafts. En block resection of the trachea and the graft with surrounding tissues was performed and sent for macroscopic and microscopic examinations. Post mortem specimens were immediately placed in 10% formaldehyde solution for preservation. The specimens were evaluated for patency, microscopic evidence of inflammation and morphologic transformation of the graft.Ten animals survived the procedure. The average operative time was 105 minutes. Between 4 to 5 tracheal rings removed in each case. One dog died 4 hours after the operation secondary to heart failure. All the animals started on diet the first post operative day. All abdominal wounds healed primarily. There was one cervical wound infection, which was healed with secondary intention. There was no clinical respiratory problem, cough or pneumonia. There was no evidence of hoarseness and all of the animals start barking a few days after the operation. There was no complication related to aortic Dacron graft. None of the animals developed claudication.At necropsy, gross examination showed a patent lumen in all specimens. The external surface was smooth and had area of depression at the site of graft. There was no abscess formation or sing of infection in the neck. There was evidence of adhesion of adjacent muscles to the trachea. There was evidence of neovascularization from the adjacent muscles to the aortic graft. Microscopic examination of H&E stained sections of grafted aorta and the anastomotic site revealed well healed anastomosis in all animals. There were areas of mild chronic inflammation, fibrosis, foreign body granuloma and squamous metaplasia in the animals sacrificed within two months of operation. In the animals sacrificed longer that 2.5 months, Pathology revealed no foreign body granuloma, minimal inflammation and ciliated columnar epithelium coverage of grafted aorta and neovascularization of aortic wall Fig , Table .Studies on prosthesis for repairing the tracheal defects have a long history beginning with Daniel's experiment in 1948. Since then, a variety of prosthesis has been used, but the results have been disappointing in a significant proportion of cases. In an extensive analysis of the literature, Grillo -12, autoIn our study, the aorta as an autogenous tissue was used to replace the trachea in a dog model. Surgical techniques for aortic segment excision and Dacron replacement with aortotracheal graft are not complicated and feasible to be done in one stage. Secondly, the aorta has no autogenous immunity-related problems and its diameter in human is comparable to the trachea. Furthermore it provides a sufficient diameter for airway patency. Due to disparity between tracheal and the aortic diameter in dogs, the aorta was transplanted to the proximal and distal tracheal and posterior membranous parts, but it appears that no such disparity is anticipated in humans. The gross and microscopic study of transplanted aortic segment showed neovascularization from adjacent tissues providing a good and resistant grafted tissue without vascular pedicle. The growth of endothelial ciliated cells over the grafted aortic segment lumen without mucous secretion and luminal stenosis is another positive point for this method in comparison with other tubular grafts such as esophagus, jejunum and collagen-conjugated meshIn conclusion, this study confirms that an autogenous aortic graft is a valuable substitute for tracheal replacement because of less antigenicity, less vascularity, and no mucous secretions or peristalsis. It also shows a progressive transformation of the vascular graft into a structure resembling the tracheal tissue. We did not have any aortic surgery related complications in our study, but it may subject the patient to potential complications of aortic surgery. The use of an arterial allograft may be more practical in human, but it remains to be demonstrated that the same tracheal neogenesis and the same long-term function can be obtained in spite of the allogeneic nature of the graft.The authors declare that they have no competing interests.FA participated in design of the study, carried out the Coordination, collecting and analyzing the data, getting grant for the study, performing the operations and post operative care, writing the manuscript.HH conceived the study, participated in design of study, supervised the study and operative procedures and reviewed the manuscript.SD participated in design of the study, anesthesia in the operative room, the operative procedures and post-operative care.NT participated in coordination for operative procedures and post operative care in animal lab.PK carried out the gross and microscopic evaluation of pathology.All authors read and approved the final manuscript.


Plasmodium berghei ANKA.Cerebral malaria (CM) is associated with high mortality and morbidity caused by a high rate of transient or persistent neurological sequelae. Studies on immunomodulatory and neuroprotective drugs as ancillary treatment in murine CM indicate promising potential. The current study was conducted to evaluate the efficacy of glatiramer acetate (GA), an immunomodulatory drug approved for the treatment of relapsing remitting multiple sclerosis, in preventing the death of C57Bl/6J mice infected with GA treatment led to a statistically significant lower risk for developing CM (57.7% versus 84.6%) in treated animals. The drug had no effect on the course of parasitaemia. The mechanism of action seems to be an immunomodulatory effect since lower IFN-gamma levels were observed in treated animals in the early course of the disease (day 4 post-infection) which also led to a lower number of brain sequestered leukocytes in treated animals. No direct neuro-protective effect such as an inhibition of apoptosis or reduction of micro-bleedings in the brain was found.These findings support the important role of the host immune response in the pathophysiology of murine CM and might lead to the development of new adjunctive treatment strategies. Plasmodium falciparum malaria is cerebral malaria (CM). It presents as a diffuse encephalopathy with alteration of consciousness, ranging from drowsiness to deep coma and is frequently accompanied by seizures [Plasmodium berghei infected mice, when applied in low dose [A major cause of morbidity and mortality of seizures . Mortaliseizures . The patseizures -5. Imporseizures ,7. Thereseizures ,9, cortiseizures ,11. Simiseizures -14. In rseizures ,16. Receseizures  and decrlow dose . The neulow dose ,20. TherGA, also known as copolymer 1, is a heterogeneous mix of polypeptides containing the four amino acids alanine, lysine, glutamic acid and tyrosine in definite ratios but with no uniform sequence with an average molecular weight of 5,000–9,000 daltons . GA was 6 parasitized red blood cells of a homologue donor, which had been infected with frozen polyclonal stocks of P. berghei ANKA. Four animals were used as non-infected control animals. The clinical severity of the disease was assessed by the SHIRPA-score primary screen on baseline, day 5 post-infection and before death [A total of 72 six to eight weeks old C57BL/6J mice  were used for this study during four subsequent experimental infections. 68 animals were infected intraperitoneally with 1 × 10re death . The prire death . HealthyFrom the day of infection, animals of the treatment group (n = 34) were injected subcutaneously daily with 100 μl Copaxone  after anesthesia with Forane  for two minutes on alternating injection sites. The control group (n = 34) was injected the same amount of saline . The course of infection was closely monitored. Core body temperature and weight were assessed daily. Parasitaemia was monitored daily by thin blood smear from tail blood. At day 4 post-infection 16 infected animals were killed to study the early course of the disease. Between day 6 and day 9 post-infection 37 of the infected mice developed signs of CM and were killed for ethical reasons as soon as their body temperature dropped to 30°C, a valid marker for imminent death . Mice wh3VO4 plus a protease inhibitor cocktail  and centrifuged at 18,500 g for 20 min at 4°C. Protein balanced samples were analysed using standard techniques. The bottom parts of the blots were probed with a monoclonal antibody directed against cleaved caspase-3  overnight at 4°C. To control and correct for equal loading, the top part of each blot was probed for alpha-tubulin  overnight at 4°C. Antibody binding was visualized using enhanced chemoluminescence reagents .Animals  were perfused with PBS for two minutes. Samples of the forebrain and brain stem with cerebellum were processed separately as described previously . The mic2O2) for 30 min and non-specific binding in blocking solution  for 1 hour at room temperature. All antibodies were diluted in blocking solution. Rabbit monoclonal antibody against activated caspase-3  was diluted 1:1,000 and permitted to bind overnight at 4°C. Biotinylated goat anti-rabbit antibody  was then applied at a dilution of 1:200 for 1 h at room temperature. Antibody binding was visualized using Vectastain ABC kit (Vector Laboratories) and diaminobenzidine as chromogen according to the manufacturer brochure and counterstained with haematoxylin. Sections without primary antibody or with primary antibody preincubated with caspase-3 blocking peptide  were equally processed to control for unspecific binding.After perfusion with 4% PFA in PBS, brains  were postfixed for six hours and cryoprotected with 30% sucrose in PBS. Frozen tissues were cut into 20 μm and 40 μm thick coronal sections on a freezing cryotome . 20 μm thick sections were mounted on Superfrost Plus slides  and air-dried. 40 μm thick sections were kept in assorter buffer and stored at 4°C. The directly mounted slides were stained with haematoxylin-eosin (H&E) according to standard protocols and used for analysis of haemorrhagic brain area. 40 μm free-floating sections were blocked for endogenous peroxidase activity  at +2, -6 ± 350 μm bregma were determined as previously described . StereolMouse serum was harvested immediately before perfusion by puncture of the right cardiac ventricle. Five different cytokines  were measured simultaneously by a commercially available cytometric bead array according to the manufacturers protocol by FACS .To test for differences in survival Kaplan-Meier curves were drawn and the treatment groups were compared using Log-rank test. Parasitaemia levels and the cumulative SHIRPA score were compared between treatment groups by repeated measures ANOVA. The relative haemorrhagic brain area, brain sequestered leukocytes and caspase-3 immunopositive brain parenchymal cells were compared between treatment groups by Wilcoxon rank sum test. Cytokine levels were compared by two-way ANOVA with time point of sampling and treatment as factors. P-values were Bonferroni-corrected for multiple comparisons. The values of IFN-gamma were logarithmically transformed to get equal variances. Calculations were done using Insightful S-Plus 6.2 , graphs were drawn by GraphPad Prism version 5.00 .Of the 68 infected animals 16 were killed on day 4 post-infection in order to study alterations in the early course of the disease. The remaining 52 animals entered the survival analysis. 37 of these mice developed signs of CM and were killed between day 5 and 9 post-infection when showing clinical signs of inevitable death . The other 15 animals survived this critical period and were killed on day 11 post-infection. 15 animals (57.7%) in the treatment group developed CM compared to 22 animals (84.6%) in the control group. Kaplan Meier curves are shown in Figure In order to assess putative differences in the clinical course of the disease, the SHIRPA score was performed on baseline, day 5, in moribund CM animals and on day 11 post-infection in animals which did not develop CM (NCM group). No significant differences were found in the respective values of the SHIRPA score between GA and vehicle treated animals Figure . There wIn order to reveal putative effects of GA treatment on the neuropathology of CM, conventional H&E staining, immunohistochemistry and western blot analysis for activated caspase-3 was performed. Qualitative inspection of H&E stained sections did not reveal differences between GA and vehicle treated animals. All moribund CM animals showed the typical neuropathology of CM . In the early course of the disease (on day 4 post-infection) and in infected animals without CM  no signs of microhaemorrhages were observed.Histopathological changes were quantified by stereological methods. In moribund CM animals the relative haemorrhagic brain area was not significantly different between GA and vehicle treated animals Figure . The relThe relative number of brain sequestered leukocytes on day 4 post-infection was not significantly different between GA and vehicle-treated animals Figure . In moriThe immuno-phenotype of animals was determined on day 4, in moribund CM animals, and on day 11 post infection in NCM animals by measuring 5 different cytokines in sera. On day 4 post-infection GA treated animals showed a significantly lower level of IFN-gamma  and other myelin associated antigens such as proteolipid protein (PLP) and myelin-oligodendrocyte glycoprotein (MOG) for this binding or even displaces proteins from the binding site . This meSequestered leukocytes are recognized to be causally related to the pathogenesis of murine CM. The onset of neurological signs and symptoms is paralleled by the sequestration of CD8+ T-lymphocytes in the brain, and depletion of these cells confers protection against CM. Accumulation of activated T cells in the brains of CM mice has been shown by different experimental approaches ,35. BesiThe neuro-protective effects of GA have been demonstrated in several animal studies. In the EAE model and in an optic-nerve injury model GA protected neurons from demyelination, axonal damage and degeneration ,42. FurtP. berghei ANKA. In general, subcutaneous treatment with GA seldom causes severe side effects [It should be noted that GA did neither change the course of parasitaemia nor the clinical course of the disease. This is of importance since a new adjunctive drug for treating severe complicated malaria must not interfere with parasite clearance through schizonticidal medication . Further effects . The mos effects . No high effects .In conclusion, the present pilot study provides direct evidence that GA, a drug which has been safely administered to patients with relapsing remitting multiple sclerosis for several years, reduces the risk for experimental cerebral malaria. The mechanism of action seems not to be neuro-protection through inhibition of caspase-3 dependent apoptosis or inhibition of parasite growth but rather immuno-modulation in the early course of the disease. Further studies are currently performed to evaluate the exact mechanisms of GA mediated immuno-modulation in CM and to determine the most effective dosing and timing of treatment. The current findings support the important role of the host immune response in the pathophysiology of murine CM and might lead to the development of new adjunctive treatment strategies.The authors declare that they have no competing interests.PL, AP, CB, AD performed all experiments. PL, GB, RH, MR, RB analyzed the data. PL, ES, RB had the idea and designed the study. All authors participated in the interpretation, and writing of the paper and read and approved the final manuscript.


Virus isolation and immunohistochemistry indicated that virus invaded the brains of guinea pigs within one day postexposure, regardless of viral strain or particle size distribution. Immunohistochemistry further demonstrated that neuroinvasion occurred through the olfactory system, followed by transneuronal spread to all regions of the brain. Olfactory bipolar neurons and neurons throughout the brain were the key viral targets. The main microscopic lesions in infected guinea pigs were neuronal necrosis, inflammation of the meninges and neuropil of the brain, and vasculitis in the brain. These results indicate that guinea pigs experimentally infected by aerosolized EEEV recapitulate several key features of fatal human infection and thus should serve as a suitable animal model for aerosol exposure to EEEV.Mice and guinea pigs were experimentally exposed to aerosols containing regionally-distinct strains (NJ1959 or ArgM) of eastern equine encephalitis virus (EEEV) at two exclusive particle size distributions. Mice were more susceptible to either strain of aerosolized EEEV than were guinea pigs; however, clinical signs indicating encephalitis were more readily observed in the guinea pigs. Lower lethality was observed in both species when EEEV was presented at the larger aerosol distribution (> 6 μm), although the differences in the median lethal dose (LD Alphavirus, family Togaviridae, that cause significant morbidity and death in infected animals and humans [Eastern equine encephalitis (EEE) virus (EEEV) are a group of positive-strand RNA viruses in the genus d humans -3. The rd humans  and expeNatural outbreaks of EEE have been reported primarily in North America; the South American varieties of EEEV appear to be less virulent in humans and animal models ,6. At a A variety of animal species have been investigated as possible disease models of human EEE. Limited pathogenicity studies using EEEV have been performed in pigs  and calvVaccines and therapeutics that protect against EEEV infection are needed to protect against the biowarfare threat posed by aerosolized EEEV. Demonstrating efficacy against aerosol exposure to EEEV would not be possible in a human clinical trial as inhalation is not a natural route of EEEV transmission. Licensure is only possible through the FDA's 'Animal Rule', which allows for the demonstration of efficacy in multiple well-characterized, relevant animal models . BecauseThe objective of this study was to initially assess the susceptibility of mice and guinea pigs to aerosolized EEEV and to describe the resulting pathology associated with experimental infection. Initially, to assess susceptibility and derive lethality estimates, groups (n = 8) of mice and guinea pigs were exposed by aerosol using either a North (NJ1959) or South American (ArgM) strains of EEEV presented at two distinctly different particle size distributions. We performed five target doses (x5) per viral strain (x2) per species (x2) per aerosol size distribution (x2) for a total of 40 individual experiments. Thereafter, groups of guinea pigs were exposed to either strain of EEEV (x2) at either aerosol size distribution (x2) to further distinguish pathology that may have resulted from multimodal aerosol exposure.ad libitum and maintained on a 12 hr light/dark cycle.Female BALB/c mice and Hartley guinea pigs of both sexes were obtained from Charles River Laboratories . The mice were approximately 19-22 g and 8-9 weeks old; the guinea pigs were 8-10 weeks old and approximately 250 g at the time of exposure. Animals were provided with rodent chow and water North American  and South American  strain of the EEEV were used in the experimentation. Viruses were propagated in cell culture as previously described . Viral tg) of 1.3 for the aerosols generated by the Collison nebulizer. The size distribution for the particles generated by the STAG was summed as being composed of distinctly 'larger' particles (> 6 μm) than the Collison-generated aerosols. Exposure concentration, expressed in plaque-forming units (PFU)/l, was determined by isokinetic sampling of the chamber with an all-glass impinger . EMEM medium with antifoam A 0.001% w/v  was used to collect medium in the impinger. 'Presented' dose was estimated by calculating the respiratory minute volume (Vm) using Guyton's formula , expressed as Vm = 2.10 × Wb 0.75 where Wb = body weight (g), based upon the average of group weights the day of exposure. The presented dose was then calculated by multiplying the estimated total volume (Vt) of experimental atmosphere inhaled by each animal (Vt = Vm × length of exposure) by the empirically determined exposure concentration (Ce) ('presented dose' = Ce × Vt).Mice were exposed to EEEV aerosols in whole-body exposure chambers housed within Class III biological safety cabinets maintained under negative pressure (-1 WC"), as previously described . The aniViral titers in the aerosol generator starting suspensions, AGI samples, tissues, and blood were determined by plaque assay as previously described. Tissues obtained at necropsy were ground with a sterile mortar and pestle and a 10% homogenate v/w solution was prepared before viral titration. Briefly, for plaque assay, samples of homogenized tissue or blood were diluted with a solution of EMEM with 5% fetal bovine serum (FBS) and gentamycin (without HEPES) added to 6-well plates containing a confluent monolayer of Vero-E6 cells (green monkey kidney cells) which were incubated at 37°C for 1 hr. Then, a 0.5% agarose overlay in 2× basal medium EMEM with Earle's salts (EBME) solution  was added, and plates were incubated at 37°C at 5% CO2 for 48 hr. Thereafter, a second overlay of saline A (SA) with 5% neutral red and 5% FBS was added, and the plates were again incubated at 37°C for 4 hr. Defined plaques  were then counted.® . A complete necropsy was conducted on each guinea pig. Heart blood samples were collected aseptically into pediatric EDTA-anticogulant evacuated tubes. Samples of cerebrum, lung, and liver were then obtained aseptically, placed in sterile centrifuge tubes, and stored at -70°C until processing for virus isolation. Additionally, a full set of tissue samples were collected for histopathology. These tissues were fixed in 10% neutral buffered formalin and stored for a minimum of 21 days within biosafety (BSL)-3 containment to ensure all virus had been deactivated before histopathology processing. Bones were decalcified in formic acid, and all tissues were trimmed, routinely processed, and embedded in paraffin. Tissue blocks were sectioned at 5-6 μm on a rotary microtome and mounted on glass slides. All tissue sections were stained by hematoxylin and eosin. In addition, duplicate sections were prepared for immunohistochemistry from the lungs, submandibular lymph nodes, haired skin, salivary glands, brain, and multiple tissues of the skull. Immunohistochemistry was performed as described previously [Guinea pigs were euthanized at the prescribed time points in the second experiment by administering an overdose of Euthasoleviously , with mo50 values were determined by probit analysis using PROC PROBIT in SAS Version 9.1.3 . Times-to-death were compared by Wilcoxon rank-sum tests. Descriptive statistics (mean ± standard deviation) were used to display the results of the tissue and blood viral titers.Dose-response curves were constructed and LDTo examine the lethality of EEEV strains when aerosolized, Hartley guinea pigs and Balb/c mice were exposed to either large (> 6 μm) or small ~1 μm) particle aerosol distributions containing the ArgM or NJ1959 strains of EEEV  although this difference was also not significant (p = 0.1057). An LD50 could not be determined for the ArgM strain at a particle size > 6 μm because mortality was not ≥ 50% in any of the dose groups.Analysis of the survival data indicated that the virulence of aerosolized EEEV was greater in mice than in guinea pigs Table . Probit 50 of 1 μm aerosol particles containing NJ1959 was over 17 times higher than what was calculated for mice  (p = 0.0098). The LD50 for the > 3 μm particles containing NJ1959 was not significantly different from the LD50 for 1 μm particles of NJ1959 (p = 0.3500) which is similar to what was observed in the mouse exposures. The LD50 for ArgM at 1 μm was half that of NJ1959  but an LD50 could not be calculated for > 6 μm ArgM particles as ≥ 50% mortality was not observed in any of the dose groups.For guinea pigs, the LD6 PFU of either NJ1959 or ArgM at 1 μm or > 6 μm particle distributions. Thereafter, the guinea pigs were euthanized on days one, two, three or four postexposure and necropsied to collect tissues for histological examination and for virus isolation. Four guinea pigs were sampled at each time point for each viral strain/particle size combination. Regardless of viral strain or particle size, virus was isolated from all four tissues examined  one day after challenge  guinea pigs at day one postexposure by positive immunohistochemistry of the olfactory mucosa, the olfactory nerves, and/or the lamina propria. Both viral strains and both particle sizes resulted in infection of the olfactory tract at this time. Affected guinea pigs had one or more foci of immunolabeled olfactory mucosa scattered throughout the nasal tract, often associated with positive olfactory nerves and lamina propria. The foci that contained EEEV antigen were without apparent morphological changes in infected cells of all cases at day one. At day two postexposure, the olfactory tracts of 15/16 (94%) guinea pigs exhibited positive immunohistochemical staining for viral antigen Figure . In indiInfection of the brains of guinea pigs was first evident by the presence of limited EEEV antigen within the lamina fibrorum and lamina glomerulosa of the olfactory bulbs of 5/16 (31%) guinea pigs at day one postexposure. Histologically, the olfactory bulbs did not exhibit abnormal changes on day one. At day two postexposure, abundant viral antigen was present throughout all layers of the olfactory bulbs of all guinea pigs and the meninges of the olfactory bulbs were minimally to mildly thickened by infiltrates of macrophages, lymphocytes, and some heterophils at this time point and at later time points Figure . There wNeurons were the primary target of EEEV in the brains of infected guinea pigs. In particular, EEEV showed tropism for medium to large neurons such as pyramidal neurons in the neocortex, Purkinje neurons, and motor neurons in the brainstem. In addition, some scattered smaller cells in the brain, possibly small neurons and/or glial cells, also contained viral antigen, as did some apparent inflammatory cells and the previously mentioned periventricular cells. Neither the strain of EEEV, nor the size of aerosol particles with which guinea pigs were infected appeared to have any effect on how rapidly virus appeared in the olfactory mucosa or the brain. However, at day two, subjectively there did appear to be slightly more antigen in both the olfactory mucosa and rostral brain of guinea pigs infected with large particle aerosols than with small particle aerosols.Minimal amounts of viral antigen were present in the lungs of some guinea pigs infected with the NJ1959 strain of EEEV Figure , but virAdditional immunohistochemistry findings included rare positive cells that appeared to be osteoblasts in the skulls of four guinea pigs, small foci of positive subgingival or periodontal connective tissue in three cases and a focus of positive odontoblastic epithelium in a single case. Otherwise, the structures of the head, including the mandibular lymph nodes, the nasal-associated lymphoid tissues, and the vomeronasal organs, did not exhibit positive immunostaining for EEEV antigen, nor was there histological evidence of viral infection of other tissues. In particular, there was no evidence of viral infection of the respiratory lining cells of the nasal tract, nor the tracheas, of any guinea pigs in the study. Evidence of EEEV infection of other tissues, including the heart, liver, spleen, kidneys, adrenal glands, stomach, small and large intestines, pancreas, urinary bladder, testes, ovaries, uterus, skin, salivary glands, thymus, bone marrow, and mediastinal and mesenteric lymph nodes, was uniformly lacking. In addition to pneumoconiosis, lesions considered to be common findings in guinea pigs were seen in some cases. Changes consistent with vitamin C deficiency were evident in the tibia and femur of most guinea pigs in the study. In several guinea pigs, rhabdomyomatosis was present in the heart and/or mild changes consistent with early segmental nephrosclerosis were present in the kidneys.This study was performed to further assess the relative infectivity of EEEV as an aerosol, to assess the comparative virulence of representative North and South American strains of EEEV when aerosolized, and to initiate characterization of rodent models of aerosol disease to be used in future testing of medical countermeasures against EEEV. In the initial exposure experiments, lethality estimates were established in both animal species, although mortality in the groups did not consistently match the increase in dose received. This variation may account for the lack of fiducial limits in some of the probit analysis. In a previous pathogenesis study using juvenile albino mice inoculated intracranially or subcutaneously with EEEV strains E-1 and P-7, a sharp dose-response curve was observed, although the doses were simply reported as an inoculating volume of 20% brain suspension homogenate from experimentally infected suckling mice, which allows no basis for comparison . Other sIn this study, the guinea pigs proved to be the better model for EEEV infection than mice, in that they developed more uniform clinical signs with an easily identified onset and lethality in the lethal dose determination. Later stages in the clinical illness  illustrated what is typically observed in encephalitic syndromes caused by neutrotropic viruses. Clinical signs were difficult to assess in mice until very late in the course of the disease. Hamsters are the alternative rodent model for studying EEEV infection. At this time, we cannot distinguish whether guinea pigs or hamsters are the superior model since both develop pathologies similar to what has been reported to humans, particularly the development of vasculitis . However50 for both strains of EEEV was higher for aerosol exposures at the larger particle size and could not be determined for the ArgM strain in mice. We pursued the serial sacrifice studies in the guinea pig model to examine the pathogenesis of aerosolized EEEV infection as a result of these initial findings in the lethality estimation experiementsIt was expected that particle size distribution would alter the virulence and pathogenesis of aerosolized EEEV. The anatomical differences in the respiratory tract of a guinea pig when compared to a mouse would affect particle deposition and subsequent initiation of disease. Larger aerosol particles of EEEV with a MMAD of > 6 μm would be expected to deposit more in the upper respiratory tract, leading to infection of the olfactory neuroepithelium and the olfactory bulb and more rapid penetration into the brain of exposed animals than would be the case for aerosol exposure to EEEV in a smaller, more highly respirable particle. Exposure to a larger particle size would be expected to lower the dose required to cause disease and potentially accelerate the progression of the infection . It was 50. Given the observed infection of the olfactory neurons by EEEV, it was not unexpected that infection of the brain was first evident as viral antigen within the olfactory bulbs and other olfactory brain structures. This was followed by apparent transneuronal spread to other parts of the brain in generally a rostral to caudal fashion, reaching the caudal brainstem and cerebellum of a few guinea pigs by day four which was the last day studied. Viral infection of the brain resulted in direct damage to neurons as well as widespread meningoencephalitis. These important pathogenetic features, infection of olfactory neuroepithelium, invasion of the olfactory brain, transneuronal spread, direct neuronal cytopathicity, and inflammation of the brain, are essentially identical to the mouse model of aerosolized VEEV, to date the best characterized animal model of aerosolized alphavirus infection [Our observation of early infection of the olfactory neuroepithelium seems to represent a cardinal feature in the pathogenesis of aerosolized EEEV in guinea pigs. In particular, EEEV infection of the so-called bipolar olfactory neurons is key, because at one 'pole' these cells have ciliary processes that extend into the air passages, where they likely contact virus, and at the opposite 'pole' they extend axons that synapse directly with neurons in the olfactory bulbs. Viruses that target the olfactory neurons therefore have a direct portal to the brain. The additional finding of early infection of olfactory neurons in guinea pigs exposed to both small- and large-particle aerosols, however, fails to explain why the guinea pigs exposed to large particle aerosols had higher LDnfection ,17-19.There were some differences between the mouse model of aerosolized VEEV and the characteristics of EEEV infection seen in our current studies with guinea pigs. Exposure to aerosolized VEEV causes massive infection of the olfactory neuroepithelium in mice, ,19 whereThere were also similarities and differences between the guinea pigs in this study and other animal models of EEEV that utilized peripheral inoculation of this virus. Key among the similarities, cutaneous inoculation of young mice ,15 and gTwo other particular features of EEEV infection of the guinea pigs in this study merit discussion. First, we did not observe tropism of EEEV for lymphoid tissues and little, if any, viral antigen within macrophages in any tissues. Studies with mice and hamsters also reported limited lymphoid tropism by EEEV ,20. ThesAn additional aim of this study was to determine the virulence and pathogenesis of a South American strain of EEEV. South American strains of EEEV are antigenically distinct from North American strains and vaccines against North American strains may not be protective . South AIn summary, mice and guinea pigs are susceptible to lethal infection by North and South American strains of EEEV. The guinea pig model of aerosolized EEEV recapitulates several key features of fatal human infection by EEEV. It also shares several important pathogenic features with aerosolized VEEV infection in mice. Thus, guinea pigs should serve as a useful animal model for aerosol exposure to alphaviruses in general, and for EEEV in particular.Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the The authors declare that they have no competing interests.CJR and DSR performed the experiments, JMH assisted with the engineering performance of the experiments. SN participated in the design of the study and performed the statistical analysis. CLW and KES performed pathological analysis. CJR conceived of the study, and DSR participated in its design and coordination. All authors read and approved the final manuscript.