The Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System Operations (AFHSC-GEIS) initiated a coordinated, multidisciplinary program to link data sets and information derived from eco-climatic remote sensing activities, ecologic niche modeling, arthropod vector, animal disease-host/reservoir, and human disease surveillance for febrile illnesses, into a predictive surveillance program that generates advisories and alerts on emerging infectious disease outbreaks. The program’s ultimate goal is pro-active public health practice through pre-event preparedness, prevention and control, and response decision-making and prioritization. This multidisciplinary program is rooted in over 10 years experience in predictive surveillance for Rift Valley fever outbreaks in Eastern Africa. The AFHSC-GEIS Rift Valley fever project is based on the identification and use of disease-emergence critical detection points as reliable signals for increased outbreak risk. The AFHSC-GEIS predictive surveillance program has formalized the Rift Valley fever project into a structured template for extending predictive surveillance capability to other Department of Defense (DoD)-priority vector- and water-borne, and zoonotic diseases and geographic areas. These include leishmaniasis, malaria, and Crimea-Congo and other viral hemorrhagic fevers in Central Asia and Africa, dengue fever in Asia and the Americas, Japanese encephalitis (JE) and chikungunya fever in Asia, and rickettsial and other tick-borne infections in the U.S., Africa and Asia. The morbidity and mortality associated with infectious disease outbreaks, which are directly or indirectly linked to ecologic or climate events and trends, pose a growing problem for global public health -3. Many The economic and human health devastation of the 1997 Rift Valley fever outbreak in Eastern Africa was not repeated in the 2006-2007 outbreak. This was largely due to the implementation of early, targeted interventions by the affected countries and the international community, prompted by AFHSC-GEIS early alerts. The result was a more contained outbreak and a quicker return to regional economic, social and political stability.To achieve its predictive surveillance goal, the AFHSC-GEIS has brought together a dynamic team of partners into an integrated, multidisciplinary program with the capability to generate and merge data to produce pre-event advisories and alerts on the emergence of disease outbreaks. Partner activities are organized into three primary predictive components: 1) satellite remote sensing and ecologic niche modeling for ecologic and climatic events, trends and characteristics that otherwise influence the potential for disease outbreaks; 2) arthropod-vector surveillance and geo-spatial mapping for characterizing vector presence, abundance, and disease transmission capability; and 3) animal-host surveillance for detecting vector and pathogen exposure events, and animal-to-animal or animal-to-human pathogen transmission. Another predictive surveillance program component, human-disease surveillance, does not serve a predictive function. Rather it provides evaluation feedback for the overall program’s meta-product, predictive information on human disease threats. The predictive surveillance program components are linked to one another by communications and data-processing channels. Program partners use the channels to integrate and analyze surveillance results from the different components for temporal and spatial relevance. This produces progressively targeted information on emerging threats.  promotes the generation of quality-controlled, reproducible data from all program participants; 2) cultivates intra- and extra-DoD predictive surveillance-related clinical and laboratory training and operational research; 3) advocates for the use of standardized surveillance and laboratory methodologies, and evaluates new or novel technologies for applicability; and 4) delineates the roles, responsibilities and mutually-supporting relationships among the AFHSC-GEIS predictive surveillance partners.The core of the program is a model that groups activities into components that conduct surveillance for critical detection points indicative of eco/climatic anomalies, and weather, disease-pathogen, vector, or animal-host events, which are known to be conducive to, and in aggregate, presage the emergence of disease outbreaks in humans. Figure  Program The AFHSC-GEIS program is expanding the use of its model to other priority vector- and water-borne and/or zoonotic diseases including: leishmaniasis, Japanese encephalitis, malaria, chikungunya fever, and hantavirus infections. The extension also covers different ecosystems such as those in southeastern Europe, central Asia, the Middle East, southeast and northeast Asia, and North America . AHFSC-GThe eco-climatic data used in predictive surveillance are derived from two sources: satellite remote sensing for the macro-level influences of climate and ecology, and ground-based measurements (ground-truths) for the micro-level results of weather and habitat conditions. The latter tend to be more precise, but geographically-localized, quantitative measurements. The former are less precise, more qualitative data that give a broad overview of eco-climate events and trends contributing to disease outbreak potential. Both are used in tandem for meaningful analysis.o C), and in the western equatorial Indian Ocean (>5o C), as its analysis’ initial critical detection point. Historical observations and expert analyses link these SSTs to excessive rainfall over Eastern Africa. When the resulting greener-than-normal conditions (vegetation growth) continue for longer than a three-month period, an ecology emerges for mosquito-presence and survival [The Global Inventory Modeling and Mapping System (GIMMS) group at the National Aeronautics and Space Administration’s (NASA) Goddard Space Flight Center monitors global-scale indicators of inter-annual climate variability, for example the El Niño Southern Oscillation (ENSO). The group performs detailed analyses of satellite-generated datasets on land surface temperature (LST),  normalizsurvival . HoweverGIMMS’ analyses result in advisories based on detections of early shifts in eco-climate trends. As these trends start to produce indicators of rising disease risk in specific geographic locations, the advisories transition to more certain alerts of threat emergence. GIMMS disseminates advisories and alerts to Federal collaborating agencies . ENM characterizes those habitat and geographic conditions suitable for maintaining disease vectors and animal hosts, and thus those areas suitable for the transmission of disease pathogens during outbreaks. Estimations of ecologic niches help investigators map precisely where a vector or host may live and thrive, providing vital information for predicting where a vector-borne or zoonotic disease may occur -20. ExamCulex tritaeniorhynchus[Cx. tritaeniorhynchus abundance, distribution and periodicity. The intended result is the identification of a putative detection point for predicting JE risk to U.S. Forces in the ROK  partners use the maximum-entropy approach, Maxent , to modeorhynchus. This apK Figure . In the http://www.mosquitomap.org) is an AFHSC-GEIS online product that incorporates ENM as a predictive surveillance tool for vector surveillance. At its core is a data-curation process that organizes and standardizes accurate mosquito collection, distribution data, and pathogen-transmission models [MosquitoMap , in 2009 they expanded to include sand fly (focus: leishmaniasis) and tick (focus: rickettsioses) surveillance.To improve Maxent accuracy and broaden its usage, investigators at the Armed Forces Research Institute of Medical Sciences (AFRIMS) adapted an RT-PCR assay for detecting JE and other flaviviruses in mosquito specimens. This improved diagnostic capability is used to increase the validity of the vector component’s identified critical detection point for JE and other flaviviruses. Also, USUHS investigators are adding a vegetation height evaluation protocol to enhance Maxent’s ecologic specificity. Both these efforts are tailored toward using the Maxent application on flavivirus threats in Southeast Asia.In anticipation, AFRIMS hosted a workshop in 2009 on ENM for 21 scientists from Thailand and the Philippines. The pathogen focus for this workshop was chikungunya virus (CHIKV), the cause of a severe, ongoing outbreak  in southern Thailand. Figure  AFRIMS eDuring 2009, USUHS trained a WRAIR entomologist to apply ENM to sand fly surveillance. This represents a first step by the predictive surveillance program to cover non-mosquito vectors of disease. In conjunction with this training, initial investigations were done to test the application of remote sensing to sand fly larval-habitat characterizations, and their use in determining critical detection points for leishmaniasis transmission in the Middle East .Leishmania spp. infection using polymerase chain reaction (PCR) techniques. This traditional surveillance work forms the basis for future ENM and predictive surveillance for leishmaniasis in that country and potentially elsewhere in sub-Saharan Africa  and the Kenya Medical Research Institute (KEMRI) conducted large-scale surveillance for sand fly presence at over 200 sites in three distinct geographic regions of Kenya. Approximately 3,500 sand flies were identified and tested for a Figure .Leishmania major-infected sand flies (species undetermined) were detected in two regions not previously known for leishmaniasis transmission: Isiolo and Lamu  in Sudan, was detected in large numbers in Isiolo and at another site, Garissa . Garissa is considered a non-endemic area for cutaneous leishmaniasis (CL) and VL [P. orientalis is rarely collected in Kenya as a whole [To date, there have been two major findings with epidemiological and predictive surveillance implications in Kenya. First, u Figure . Second,) and VL -36. P. o a whole .Interestingly, USAMRU-K investigators are receiving reports from the Wajir area in northeast Kenya on possible VL cases among inhabitants and refugees from Ethiopia and Somalia. This province is not traditionally considered endemic for leishmaniasis either. However, the area did undergo drought conditions after the 2006-2007 El Niño rains; and similar reports of leishmaniasis were made during the aftermath of the El Niño events of 1997 and 2000-2001  . TherefoAmblyomma americanum, Dermacentor variabilis, Amblyomma maculatum). The ticks were tested for rickettsiae (Borrelia lonestari and Ehrlichia chaffeensis), using quantitative real-time PCR (qPCR) assays and multilocus sequence typing (MLST).Tick surveillance is also conducted in the predictive surveillance program’s vector component. As an initiation point, the program is using rickettsiae as the pathogen focus. During 2009 at Fort Eustis, Virginia, partners from the Naval Medical Research Center (NMRC) collected three species of ticks known to parasitize humans  of the pools had positive reactions to Rickettsia amblyommii (unknown pathogenicity), two (6.1 percent) for B. lonestari (unknown pathogenicity), and one (3 percent) for E. chaffeensis (human monocytic ehrlichiosis). All six pools  of D. variabilis were negative for evidence of rickettsiae. Rickettsia parkeri, a newly recognized pathogen, [A. maculatum tick. This is significant because until 2002, all confirmed cases of tick-borne spotted fever in North America were attributed to only one pathogen, Rickettsia rickettsii, the cause of Rocky Mountain spotted fever. Previously, tick-borne rickettsiae other than R. rickettsii were considered non-pathogenic in the United States. Between 2002 and 2006, R. parkeri infection was detected in six cases (two DoD beneficiaries) in the southeast United States. This detection and subsequent investigations of R. parkeri infection in the U.S. are being used to build future predictive surveillance capability on this and other tick-associated diseases.Researchers tested 108 athogen,  was founst Hospital with eschars typical for spotted fever group (SFG) and scrub typhus group (STG) rickettsial infections. This observation prompted a serology study in 2005 which resulted in the first evidence that SFG rickettsial infections are associated with deployments of U.S. military personnel to Korea, 181 seropositive of 8,918 tested (2 percent).Overseas, an astute AFHSC-GEIS partner in the ROK noted that in 2004 two service members presented at the 121While it is generally accepted that eco-climate trends and events can influence disease transmission from animals to humans ,41, giveIn Kenya, USAMRU-K discovered hantaviruses in live rodents from Marigat and Garissa Figure . These vIn the ROK, U.S. and Korean partners are conducting small mammal surveillance to elucidate hantavirus exposure-risk at U.S. field training sites. Recently, these investigators discovered three new hantaviruses  -44.R. felis and another SFG Rickettsia spp. were found associated with 208 fleas (Xenopsylla cheopis) from these rodents. Similar studies were conducted with partners in the ROK [R. parkeri {Tidewater spotted fever}, Candidatus, Rickettsia andeanae {unknown pathogenicity}, and R. felis {flea-borne spotted fever}).To complement the vector component work on rickettsial infections, Naval Medical Research Unit No. 2, Jakarta, Indonesia (NAMRU-2) has found rickettsiae in small peri-domestic mammals on the islands of Java, Kalimantan, Sulawesi and Sumatra in Indonesia. In a total of 491 animals , 26 percent were seropositive for SFG rickettsiae, 14 percent for typhus group, and another 10 percent for STG rickettsiae. Interestingly,  the ROK  and in PAs the AFHSC-GEIS predictive surveillance model is framed, the human component comprises two aspects of human-based surveillance and analysis. The first is human disease and case detections from laboratory diagnosis or syndromic surveillance. This human disease surveillance is described elsewhere  and willThe second aspect, and the one that is integral to the predictive surveillance model itself, is the identification and characterization of those specific human factors that can serve as critical detection points for human-disease outbreak emergence. As an initial pilot study (using a directly-transmitted disease - cholera), the Johns Hopkins University Applied Physics Laboratory (JHU/APL) assessed the demographic, economic, environmental, and climatic variables associated with outbreaks in Africa during 1995-2005. The study’s putative contributing factors for outbreaks included qualitative and quantitative sanitation and water source contamination, rainfall, and water management  factors, human housing density and quality , and human migration and living-practices associated with displaced persons or refugees. This study showed good correlation between these factors and cholera outbreaks. It also showed that the occurrence of a cholera outbreak in a neighboring province during the previous month could also be a predictive factor . This stAs each of the above components strives to identify the critical detection point(s) which signal(s) a potential for pathogen transmission, it is understood that the predictive surveillance model’s underpinning is pathogen presence or absence in a geographic area, not the intrinsic behavior of the pathogen itself. This necessitates the inclusion of accurate diagnostic testing capability in the program’s activities. During 2009, this aspect of the program was best exemplified by work done by NMRC on rickettsiae detection and characterization.Rickettsia felis (flea-borne spotted fever) in febrile patients from northeastern Kenya. By developing and providing an Enzyme-linked Immunosorbent Assay (ELISA) test specific for SFG rickettsiae antibodies against Rickettsia conorii, NAMRU-2 confirmed the presence of SFG rickettsioses among fever patients in Cambodia .After developing and producing rickettsiae diagnostic assays, NMRC used them to test arthropod vectors, animal hosts and specimens from human cases for rickettsial infections . NMRC haNAMRU-2 has also performed SFG rickettsiae-specific ELISA testing on 368 sera collected from residents of Jakarta, Indonesia. In this work, NAMRU-2 found SFG rickettsiae-specific antibodies in 60 serum samples (16.3 percent), and of these, five paired samples showed a four-fold rise in titer indicating acute SFG rickettsiosis. Likewise, NAMRU-2 was able to test pooled ectoparasites from rodents collected in Jakarta, and found 2/40 (5 percent) positive for SFG rickettsial DNA.Through the predictive surveillance program, NMRC will continue to provide DoD investigators with technical assistance and assays for detecting rickettsial diseases  that present emerging and re-emerging risk to DoD military, civilian, and family members throughout the world.To ensure reliability and validity, the AFHSC-GEIS program is promoting consensus on and the establishment of dataset standards for the program’s component activities. Dataset standardization is necessary for conducting meta-analyses on the results of program activities, and merging them into meaningful information for advisories, alerts and predictions. For example, precise taxonomic and geo-referenced collection data are essential for understanding vector and host bio-geography, ecology and distributions, and the impact of environmental changes on them. The data are essential for determining pathogen transmission factors associated with outbreaks. Accurately geo-referenced vector collection data must be matched spatially and temporally with remote sensing data of an appropriate resolution to answer questions about the environmental determinants of vector-borne disease distribution. While the methodology of collecting datasets may vary according to the instruments used, the end results, the language and content of the datasets must be concordant in order to facilitate a seamless progression of surveillance results between AFHSC-GEIS partners and program components. Partners at WRAIR-WRBU have published recommendations on mosquito collection data standards, and these are being considered as the standard operating procedure (SOP) for mosquito collections sponsored by AFHSC-GEIS . SimilarLooking to the future, the AFHSC-GEIS predictive surveillance program in its entirety, its management framework of the AFHSC-GEIS Predictive Surveillance Steering Committee, and its scientific model that provides direction to program surveillance and analysis activities, will continue to support the detection and reporting of emerging vector- and water-borne and zoonotic disease outbreaks of importance to DoD.Under AFHSC-GEIS leadership, the program will refine and expand its model of disease outbreak emergence surveillance. It will build and elaborate upon those component activities that optimize collaborations between partners. It will continue to define those critical detection points that represent the model’s signal that an outbreak is emerging. The program will continue to advocate for focused, pre-emptive public health action to prevent or mitigate human morbidity and mortality, as well as the destabilizing effects of infectious disease outbreaks.In the near term, the predictive surveillance program is expanding upon its initial Rift Valley fever success. It will continue to characterize and validate identified eco-climate anomalies for predictive surveillance application under different ecosystem and habitat conditions. Partners will continue to identify disease-associated vectors and hosts with demonstrated periodic or cyclic disease-transmission pathways that are plausibly associated with eco-climatic events and trends. Partners will investigate vector- and water-borne and zoonotic diseases for which the program’s model might be applicable, and most importantly, which present operational risks to DoD force. This expansion is now underway with the program’s activities on JE, leishmaniasis, hantavirus and chikungunya fever. The program is initiating pilot activities on Crimea-Congo hemorrhagic fever, Ebola and other viral hemorrhagic fever outbreaks, and malaria.By continuing efforts in strengthening pathogen detection capabilities, program components are improving the sensitivity and specificity of the model’s critical detection points. As the program expands, partners will learn more about the model’s capabilities and determine where, for what and under what conditions the model does or does not apply. This will necessitate an incremental growth for the program, subjecting model usage to continuous quality assurance and applicability assessments. As the program matures, AFHSC-GEIS will facilitate the appropriate transfer of the components’ operations to those partner organizations best suited to ensure sustainable, quality-assured predictive surveillance.The success of predictive surveillance will depend on the recognition that it is founded on a meta-system of different surveillance activities, linked by communications, coordination and collaboration between multiple, diverse disciplines, and producing advisories and alerts, which have progressively more certainty over the course of an outbreak’s emergence. No single component can provide sufficient information for accurate predictions. Just because we detect an El Niño event, does not mean that a disease outbreak will occur. Similarly, if a vector is present in a locality, it does not mean that the pathogen is present or that disease will be transmitted to humans. Human activities may permit, exacerbate, or prevent human exposure to the risk of disease transmission. It is only when all relevant components of the predictive surveillance model are applied diligently and with scientific rigor to the synthesis of a complete agent-vector-host-environmental scenario, that the program will produce reliable advisories, alerts and predictions.The role of AFHSC-GEIS in the predictive surveillance program as a whole is to provide an agile, investigative platform for continuously testing different scenarios, combinations of geographic areas, vector- and host-species characteristics, and pathogens to facilitate the development of a stable of predictive surveillance models for an array of different infectious disease outbreak scenarios. In the end, however, even once given, a prediction, advisory, or alert cannot prevent outbreaks. It can only inform the decision-making process for timely, effective public health action.The authors declare no competing interests.


While most allergic responses to food are directed against protein epitopes and occur within 30 minutes of ingesting the allergen, recent studies suggest that delayed reactions may occur, sometimes mediated by IgE antibodies directed against carbohydrate moieties. The objective of this review is to summarize the clinical features and management of delayed hypersensitivity reactions to mammalian meat mediated by IgE antibodies to galactose-alpha 1,3-galactose , an oligosaccharide.A PubMed search was conducted with MeSH terms: galactosyl- galactose, oligosaccharides, cetuximab, allergy/hypersensitivity, and anaphylaxis. Reported cases with alpha-gal-mediated reactions were reviewed. This research study was approved by the Institutional Review Board of East Tennessee State University.Thirty-two cases of adults presenting with red-meat induced allergy thought to be related to oligosaccharides have been reported in the literature so far, making this a rare and evolving syndrome. Most of these patients demonstrated delayed reactions to beef, as was seen in the case reported by us in this manuscript. IgE specific to alpha-gal was identified in most patients with variable response to skin testing with beef and pork. Inhibition studies in some cases showed that the IgE antibodies to beef were directed towards alpha-gal in the meat rather than the protein. The patients often reported history of tick bites, the significance of which is unclear at present. Reactions to cetuximab, a monoclonal antibody, are mediated by a similar mechanism, with IgE antibodies directed against an alpha-gal moiety incorporated in the drug structure.Alpha-gal is an oligosaccharide recently incriminated in delayed anaphylactic reactions to mammalian meats such as to beef, pork, and lamb. It appears that anaphylactic reactions to the anti-cancer biological agent, cetuximab, may be linked mechanistically to the same process. More studies are required to understand the underlying molecular basis for these delayed reactions in specific, and their broader implications for host defense in general. Food allergy remains a well-recognized problem that affects people of different ages and can alter their quality of life ,2. Its pAmong food allergies, hypersensitivity to red meat is less common -22. It wIt was assumed that reactions to mammalian meats would be immediate, and due to IgE directed against specific protein allergens such as bovine serum albumin (BSA) ,27,42-46Alpha-gal Figure  is an olExposure to food allergens  results in the generation of IgG antibodies from B cells and hence development of immune tolerance. In predisposed individuals, due to possible genetic/environmental factors , exposure of antigen presenting cells to alpha-gal leads to Th2 activation and induction of interleukins, leading to IgE formation by B cells. This culminates in mast cell activation, eosinophilia, and the full gamut of IgE-mediated hypersensitivity reactions characterized by urticaria, angioedema, and progression to systemic anaphylaxis in some patients ,57,58.http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/2004/ucm108244.htm) [http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/2006/ucm108609.htm). Since 2002, cetuximab has induced severe hypersensitivity reactions [Cetuximab is a recombinant chimeric epidermal growth factor monoclonal antibody approved for treatment of metastatic colorectal and head and neck cancer ,59-61. Ieactions ,68-70 theactions -73. In 2eactions ,68  can be classified as IgE and non-IgE mediated reactions, and can present in the form of skin, pulmonary, or cardiac manifestations -79. Non-As mentioned earlier, Chung et al. identified IgE antibodies to alpha-gal in patients presenting with severe infusion reactions to cetuximab . In thisEach cetuximab molecule contains two alpha-gal epitopes that can cross-link the high affinity receptor for IgE (FcεRI) on mast cells . IgE croThe clinical presentation of alpha-gal-related red meat allergy is similar to other food allergies, but has many unique characteristics , commonThe bite of the lone star tick is associated with the development of STARI   is associated with development of meat-induced allergy [Ixodes holocyclus [Ixodes ricinus [In other areas of the world, history of tick bites (different than  allergy ,53. In alocyclus , and is locyclus -98. In Slocyclus . Althoug ricinus . Cases wAnother interesting relationship between the areas where alpha-gal allergy is reported in the United States is the obesity rate. The recently reported obesity rates from the Center of Disease Control and Prevention for Tennessee, Arkansas, and North Carolina were higher than those in California and Massachusetts, areas where alpha-gal allergy is uncommon . This raThe mechanism behind the delayed presentation of symptoms after red meat ingestion in patients with IgE to alpha-gal is still undetermined. A possible explanation suggested by Commins et al. proposes this delay to the time needed for red meat to be digested and presented to circulation . Lipid aAlpha-gal is known to be abundantly present on glycolipids and glycoproteins of non-primate mammals, including the red meat from beef, pork, and lamb ,50. GlycThe delay in appearance of symptoms related to alpha-gal allergy makes it difficult for patients and physicians to identify the trigger . If suchAlpha-gal allergy is a new and evolving syndrome related to oligosaccharides, rather than protein, in red meat. The IgE mediated response to alpha-gal tends to occur several hours after antigen exposure. This unique presentation constitutes a challenge to both patients and physicians, making detailed history very important if suspected. Reactions to cetuximab seem to be mediated by an identical mechanism. More studies are required about this unique syndrome, including an explanation for the mechanism of delay in presentation and the possible roles obesity and tick bites play in the predisposition for the disorder.Alpha-gal: Galactose-alpha 1,3-galactose; BSA: Bovine serum albumin; SPT: Skin-prick test; FEIA: Fluorescence enzyme immunoassay; VLDLs: Very-low density lipoproteins; FDEIA: Food-dependent exercise-induced anaphylaxis; OSA: Ovine serum albumin.The authors declare that they have no competing interests.HS reviewed the literature, generated references, organized the manuscript, and illustrated the figures. SE assisted with manuscript review and corrections. AN assisted in section involving mechanism of delay and role of chylomicrons. SA assisted in section involving mechanism of delay and role of chylomicrons. GK organized the manuscript, edited figures and tables, assisted in discussion, generated references, and participated in the editing and final approval of the manuscript. All authors read and approved the final manuscript.Table 1. Common food allergens.Click here for fileTable 2. Meat Allergy Types and Features.Click here for fileTable 3. Diagnostic features of alpha-gal-related food allergy.Click here for fileTable 4. Summary of reported cases with alpha-gal allergy in USA and Europe.Click here for fileTable 5. Acute Management and Prevention of Anaphylaxis*.Click here for file


After witnessing an episode of poor injection safety in large numbers of children in a rural under-resourced hospital in Uganda, we briefly review our own experience and that of others in investigating HIV infection in children considered unlikely to be through commonly identified routes such as vertical transmission, sexual abuse or blood transfusion. In the majority of cases, parents are HIV uninfected. The cumulative experience suggests that the problem is real, but with relatively low frequency. Vertical transmission is the major route for HIV to children. However, factors such as poor injection safety, undocumented surrogate breast feeding, an HIV-infected adult feeding premasticated food to a weaning toddler, poor hygienic practice in the home and using unsterilised equipment for minor surgical or traditional procedures are of cumulative concern. Major strides have been made to reduce the rate of MTCT . Howeverzambique .The ward was staffed by dedicated healthcare workers, but there were few nurses looking after a huge number of children in an overcrowded paediatric ward. Resources were scarce. Children were not provided with food. Since cots were limited, parents  mostly slept underneath their children's cots or children slept with their parents on the ground. Provision for hand washing was scarce. There was a single working tap outside the ward, but without soap. There was no alcohol hand disinfectant. Children, accompanied by their parents, were lined up to receive intravenous (IV) medication. All medications were withdrawn from multi-dose vials, mostly containing quinine, ampicillin or cloxacillin. Children had IV lines or locks for IV access and were injected with the same syringe (and needle in most instances). Needles were switched when moving from one vial to the next. The syringe was only discarded after all medications had been given. The nurse giving the IV medicine guided the needle into the IV hub with her ungloved finger, seemingly unaware of the grave infection risk.One of us (BJM) witnessed poor injection safety in a paediatric ward in a hospital while on a field trip in a malaria-endemic region of rural Uganda. The episode seemed representative of the situation in large parts of sub-Saharan Africa:This incident prompted us to review our own and others’ experience of possible non-vertical, non-sexual HIV in children, summarized in We first became aware of this problem in 1998 when asked to investigate three children with HIV-uninfected parents at a regional hospital near Cape Town, South Africa. We confirmed the HIV-negative status of parents and siblings in all three families. However, subsequently we documented HIV infection in a previously uninfected sibling. The likely explanation was that the HIV-infected index patient developed epistaxis together with chronic otorrhea when the younger brother had herpes stomatitis. The siblings slept in the same bed. Relatedness of HIV was confirmed by phylogenetic linkage studies . We wereWe reported 14 cases from the registry in 2004 . All hadSince 2004, we reported an additional 10 children with probable non-vertical, non-sexual HIV infection . Nine haet al. reported an epidemiological study of 6991 children and their mothers in rural southwest Uganda. The prevalence of HIV in children above 18 months of age, when antibody detection is reliable for infection status, was 0.37% (26 of 6981). Depending on the level of certainty, between one and three children had non-vertical, non-sexual transmission. Therapeutic injections, including blood transfusions were more likely in children probably infected through MTCT, suggesting that these children were sicker rather than a potential for exposure to contaminated blood or equipment [et al. described 22 HIV-infected children with HIV-uninfected parents, comprising 4.8% of their clinic cohort in Maputo, Mozambique. They identified blood transfusions as a possible risk factor in 12 of the 22 children [There is conflicting evidence about the scale of this problem. For example, Biraro quipment . SimilarThe extent of non-vertical, non-sexual infections could be far higher in some areas. For example, in an analysis of the 2006–2007 Swaziland Demographic and Health Survey, 11 out of 50 HIV-infected children had HIV-seronegative mothers. The same authors also identified eight children in Kenya with HIV-seronegative mothers through seeking referrals from doctors and nurses .et al. [In France, Frange et al.  describeet al. . TransfuThe first report implicating premastication as a potential risk factor for HIV transmission  was follet al. [Inadequate knowledge of blood-borne virus transmission risk seems prevalent among health care workers and the general population. Infection control practices are often poorly implemented and remain undervalued in many HIV-endemic settings. In particular, unsafe injection practices are a major problem. In 2000, Hutin et al.  estimateet al. .et al. reported that 1.1% of 5446 respondents re-used a syringe to enter a multi-dose vial and then saved that vial for another patient [et al. recently reported an outbreak of hepatitis C virus and human T cell lymphotropic virus-1 (HTLV-1) in Equatorial Africa linked to chemoprophylaxis administered by intramuscular injection for trypanosomiasis prior to 1951. They also hypothesize that widespread unsafe injection practices could have facilitated the initial spread of HIV-1 [Surprisingly, there are numerous reports of blood-borne virus transmission in hospital settings through poor infection control in resource-rich settings. Re-use of syringes , multi-u patient . The dat patient , and 44% patient . Pépin eof HIV-1 . The samof HIV-1 ,38. ThisAs an extreme example, large iatrogenic outbreaks of HIV have been associated with non-sterile medical equipment usage in children from Kazakhstan and Uzbekistan ,40. In aThe WHO has developed a toolkit for assessing injection safety . The tooet al., from Australia, reported probable spread between two sisters who shared a bathroom and sometimes also the same razor to shave body hair and from a male with psoriasis to his mother, who provided topical care without gloves [Apart from our own case report , intra-ht gloves . In a ret gloves .There appears to be a low but real risk of non-vertical, non-sexual HIV transmission (and other blood born viruses) to children. The need to decrease risks associated with hospitalization and medical care is well recognized , but ver


Fibromyalgia (FM), characterized by wide-spread diffuse pain and sensory abnormalities, is associated with elevated indices of distress and pain-related catastrophizing compared to both pain-free samples and those with chronic pain conditions. Catastrophizing is a pervasive negative mental set, and is a strong predictor of negative pain-related outcomes such as clinical pain intensity, and physical disability. Situational catastrophizing, measured in the context of experimentally-induced pain, is strongly related to enhanced pain sensitivity, a core aspect of the pathophysiology of fibromyalgia. However, little is known regarding the temporal course of the association between catastrophizing and pain-related "outcomes". Most studies involve only static assessments of pain and catastrophizing at a single time point, which provides little insight into the direction of the observed associations. We sought to investigate the temporal relationships between catastrophizing and indices of both clinical pain (substudy 1) and experimentally-induced pain (substudy 2) in a larger randomized controlled longitudinal trial.Fifty-seven patients with FM completed catastrophizing, depression, and pain questionnaires as well as laboratory cold pressor pain testing at baseline, post-intervention and three month follow-up during a lifestyle physical activity study. Cross-lagged panel analyses were used to address these temporal relationships.In substudy 1, analyses revealed that pre-to-post changes in dispositional catastrophizing ratings prospectively accounted for unique variance in subsequent post-to-follow-up changes in clinical pain ratings (p = 0.005), while pre-to-post changes in pain ratings did not account for unique variance in post-to-follow-up changes in catastrophizing ratings. An identical pattern was observed experimentally in substudy 2, with pre-to-post changes in situational catastrophizing ratings prospectively accounting for unique variance in subsequent post-to-follow-up changes in experimental pain ratings (p = 0.014), while pre-to-post changes in pain ratings did not account for unique variance in post-to-follow-up changes in catastrophizing ratings. Specifically, initial alterations in catastrophizing were associated with subsequent alterations in clinical and experimentally induced pain. Controlling for levels of depression did not affect the results.These findings provide empirical evidence that catastrophizing processes might precede and contribute to subsequent alterations in the pain experience for FM patients.NCT00383084.clinicaltrials.gov:  Catastrophizing, a set of negative emotional and cognitive processes, is widely recognized as an important factor in amplifying chronic pain . DisposiSituational catastrophizing measured during or immediately following laboratory pain procedures, is also strongly related to experimental pain ratings . Our groFM patients were recruited for participation through the Johns Hopkins Arthritis Center, affiliated Johns Hopkins Rheumatology clinics, by newspaper advertisements, and clinical trial recruitment websites, including clinicaltrials.gov. Substudy 1 included a total sample size of 57 participants for analyses of clinical pain. Eight participants elected not to complete follow-up cold pressor testing, thus 49 are included in substudy 2, the experimental pain analyses. Mean age of the sample was 48.1 years SD 11.5), and the group was predominantly female (95%) and non-Hispanic white (90%). The mean duration of FM was 7.4 years (SD 6.6). Those excluded from the experimental analyses were not significantly different from the larger group in any demographic category. Major inclusion criteria included age ≥ 18 years and who met American College of Rheumatology diagnostic criteria for FM .5, and t,13 for aParticipants were randomized to either a lifestyle physical activity (LPA) intervention group or a fibromyalgia education (FME) control group. LPA included six 60-minute group sessions over 12 weeks; sessions addressed FM-specific challenges to becoming more physically active, with a goal of accumulating 30 minutes of self-selected moderate-intensity physical activity five to seven days each week. The FME group met monthly for three months and included educational components and social support  where participants rated their current level of pain, ranging from 0 (no pain) to 100 (worst pain imaginable).Participants completed the Pain Catastrophizing Scale (PCS)  at each Cold pressor pain was assessed by having participants immerse their nondominant hand up to the wrist in a 4°C water bath. Standardized instructions informed participants that they should maintain their hand in the water bath for 30 seconds; however if the pain became intolerable, participants were told that they could remove their hand at any time. Subjects were prompted to rate the intensity of the cold pressor pain using a 0 to 100 VAS. This procedure was conducted twice to ensure consistent readings. Pain ratings were averaged across the two trials for all subsequent analyses.An adaptation of the Pain Catastrophizing Scale  used forAnalysis of variance (ANOVA) was conducted to determine any overall effect of group (LPA vs. FME) on the variables of interest. Clinical pain ratings were used, and mean cold pressor pain ratings and summed dispositional and situational catastrophizing were calculated for each of the three time points. Zero-order correlations between both catastrophizing measures and pain measures at all three time pointes were computed. Three-factor repeated-measures ANOVA the factors being assessments made before treatment (Pre), immediately following 12-week treatment (Post), and at follow-up 3-months post-treatment (FU), were conducted to determine whether changes were observed from Pre assessments to Post and FU assessment epochs for dispositional and situational catastrophizing and clinical and cold pressor pain ratings. We computed standardized residualized change scores to index Pre-to-Post and Post-to-FU changes in each measure. We chose to use residualized change scores instead of simple change scores because of problems of dependence between change and Pre values encountered with use of the latter . The useP = 0.01). As displayed in Table r 0.79; P < 0.001), as were situational catastrophizing . Clinical pain was only significantly correlated at Pre and FU , while cold pressor pain ratings were correlated with each other at each time point . The average of the three concurrent correlations between PCS and SPCS (mean r 0.26), between PCS and clinical pain (mean r 0.26) and PCS and cold pressor pain (mean r 0.14) was inconsistent. SPCS was correlated with clinical pain (mean r 0.08) and cold pressor pain (mean r 0.37), again with inconsistent degrees of significance. On average, clinical pain was marginally correlated with cold pressor pain (mean r 0.14).No group differences emerged by condition for any variable, with the exception of mean Post pain VAS score  decreased significantly over time (from Pre-to-Post and Pre-to-FU). However, situational catastrophizing, clinical pain and cold pressor pain ratings did not substantially change over time.As shown in Table Correlations among standardized residual change scores are presented in Table With Post-to-FU change in dispositional catastrophizing as the criterion variable, change in dispositional catastrophizing from Pre-to-Post and change in pain from Post-to-FU accounted for 14% of the variance in the initial step of the regression. In the second step, Pre-to-Post change in pain accounted for only 0.1% of the variance in Post-to-FU change in catastrophizing  from Pre-to-Post assessment was associated with (subsequent) changes in Post-to-FU clinical pain. The same relationship was observed for situational catastrophizing . These findings were observed even when controlling for Post-to-FU changes in catastrophizing and Pre-to-Post changes in pain . In contrast, changes in clinical and experimental pain from Pre-to-Post were not associated with later changes in Post-to-FU catastrophizing. These results provide additional evidence that changes in catastrophizing might precede changes in pain response. Our group previously demonstrated this association in healthy participants undergoing laboratory capsaicin-induced pain . This apConventionally measured catastrophizing is thought to reflect a dispositional trait that appears to be relatively stable over time in the absence of intervention -22. MeasP < 0.001) [A growing collection of studies have shown that catastrophizing is linked to a number of pain-related symptoms in FM patients, including pain severity, disability and tender point counts . Catastr< 0.001) .Several studies have found no association between dispositional and situational catastrophizing ,16. UnliRecent studies have suggested that the pain-reducing effects of a variety of analgesic treatments, including non-psychological interventions, are due partially to their effects on cognitive-emotional processes such as catastrophizing. For example, exercise- and activity-based physical therapy interventions are effective in reducing chronic low back pain, and their analgesic effects are substantially mediated by the decreases they produce in catastrophizing ,36. The In terms of mechanisms by which early changes in catastrophizing shape later changes in pain, some prior reports highlight compliance and willingness to engage in physical activity. Helplessness, a key component of catastrophizing, correlates with less effective compliance with treatment recommendations ,38, and It is interesting to speculate that treatments that specifically reduce catastrophizing might augment interventions such as LPA. Several studies indicate that cognitive behavioral therapy (CBT) and related interventions might reduce catastrophizing, with positive effects on later pain and functional outcomes. For example, Riddle and colleagues studied total knee replacement patients, selectively recruiting a highly distressed, high-catastrophizing group . FollowiSeveral limitations should be considered in the context of this work. First, this constitutes a relatively small sample of patients, given that all included here had to complete all time points. Second, these data are pooled from two treatment groups  from one larger randomized controlled trial. While no differences were observed between groups at the final treatment visit, we are unable to determine the influences of treatment over the course of the study. Thus, it is unclear how treatment interacted with (reduced) catastrophizing and pain between and within groups. Dispositional catastrophizing was reduced over time, while situational catastrophizing remained relatively constant. However, fluctuations in situational catastrophizing significantly predicted alterations in cold pressor pain responses over time, despite this relative stability. This may be explained by variation in individual catastrophizing responses driving the influence over pain reporting ,43. In aThese data are novel as few longitudinal studies have examined changes in catastrophizing and pain in FM patients. In addition, this study extends our previous work ,8 and thANOVA: analyses of variance; CBT: cognitive behavioral therapy; FM: fibromyalgia; FME: fibromyalgia education; LPA: lifestyle physical activity; PCS: Pain Catastrophizing Scale; SPCS: Situation-Specific Pain Catastrophizing Scale; VAS: visual analogue scale.The authors declare that they have no competing interests.LC, LM, SB, and MS contributed to the collection, inputting and scoring/interpretation of data. KF and RE designed the study and participated in the oversight of the study. VM and RE contributed to the analyses, interpretation and writing. CC wrote the initial draft of the manuscript and conducted the final analyses. All authors contributed to the finalization and critical assessment of the manuscript and have given final approval of this version.


Arthropod-borne viral encephalitis of diverse origins shows similar clinical symptoms, histopathology and magnetic resonance imaging, indicating that the patho mechanisms may be similar. There is no specific therapy to date. However, vaccination remains the best prophylaxis against a selected few. Regardless of these shortcomings, there are an increasing number of case reports that successfully treat arboviral encephalitis with high doses of intravenous immunoglobulins.To our knowledge, high dose intravenous immunoglobulin has not been tested systematically for treating severe cases of tick-borne encephalitis. Antibody-dependent enhancement has been suspected, but not proven, in several juvenile cases of tick-borne encephalitis. Although antibody-dependent enhancement during secondary infection with dengue virus has been documented, no adverse effects were noticed in a controlled study of high dose intravenous immunoglobulin therapy for dengue-associated thrombocytopenia. The inflammation-dampening therapeutic effects of generic high dose intravenous immunoglobulins may override the antibody-dependent enhancement effects that are potentially induced by cross-reactive antibodies or by virus-specific antibodies at sub-neutralizing levels.Analogous to the increasing number of case reports on the successful treatment of other arboviral encephalitides with high dose intravenous immunoglobulins, we postulate whether it may be possible to also treat severe cases of tick-borne encephalitis with high dose intravenous immunoglobulins as early in the course of the disease as possible. Flaviviridae family (for review see [Ixodes (I.) ricinus, while the Eastern subtypes are mainly transmitted by the tick species I. persulcatus from rodents and other small mammals. Vaccination is highly effective in preventing TBE disease and is recommended for residents exposed to TBEV within the endemic areas [Together with other prominent human pathogenic arthropod-borne (arbo) viruses, such as yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), West-Nile virus (WNV) and other less known relatives, the tick-borne encephalitis virus (TBEV) belongs to the view see ). Flavivic areas . Firstlyic areas ,3. Sinceic areas . In eldeic areas ,5). It hic areas -8, as itic areas , it has ic areas .Widespread lesions in the CNS may involve gray matter and leptomeninges of the brain stem, medulla oblongata, nuclei, cerebellum and spinal cord . A pr+ T-cells to infected SCID mice significantly shortened their survival, while the transfer of CD4+ T-cells prolonged their survival. The viral load was the same in normal mouse strains and in CD8-knockout mice, while it was higher in SCID mice. The histological brain lesions were more moderate in immune suppressed mouse strains than in immunocompetent mice. The inflammatory infiltrates around meningeal vessels consisted mainly of CD8+ T-cells, and contained also histiocytes, but little CD4+ T-cells. These experiments proved a key role for the CD8+ T-cells in the pathology of TBE, and that TBE is essentially an immunopathological disease [+ T-cells were the main culprits for the immune damage caused by WNV, while CD4+ T-cells prolonged the survival of WNV infected mice [+ T-cells, WNV load was lower in immunocompetent mice. Both in TBEV and WNV encephalitis, CD8+ T-cells were required for the eventual clearance of WNV from the organism and for mouse survival [The mechanisms by which TBEV causes encephalitis are not completely understood, but a composite of direct cytolytic viral damage and of considerable immune pathology is likely. Mouse models of TBE disease demonstrated that immune pathology contributed significantly to the neurological damage in TBE . On the  disease . Moreove disease . The CNSted mice ,17. Contsurvival ,18. Thissurvival ,19. Thussurvival .There is no specific treatment currently available for TBEV disease. Since immune modulation by tetracycline was helpful, immunomodulatory therapy may be the way to go . Ideal tThe neuropathological and neuroradiological findings of various arboviral encephalitides are similar in nature and therefore non-specific ,25. TherA 54 year old man had a severe and complicated TBE infection, after a complete vaccination against TBE with two boosters. He received the last booster 39 months before his disease. After two weeks the patient developed sinus tachycardia, hypertonia and gastrointestinal tract dysfunction. These symptoms and a fixed heart rate were taken as signs of autonomic failure. Late after disease onset, i.e. from day 20 to 24 of his illness, the patient received IVIG at a dose of 0.4 grams per kilogram body weight each day. While the dysautonomic symptoms improved and cardiac arrhythmia disappeared, the other neurologic symptoms did not change, and a post-encephalitic syndrome persisted that forced the patient into early retirement . In thisJEV is the most common cause of mosquito-borne flaviviral encephalitis in Asia. About 30,000 to 50,000 cases are reported annually. Contrary to TBE, mortality rate is high in the range of 25 to 30%. Sequelae are observed in about 50% of those who survive ,29. A 49In addition to hypotension, respiratory failure and general meningoencephalitic symptoms, a 64-year-old man with JE had prominent signs of Parkinsonism. MRI scans showed T2-weighted hyper intensities in the thalamus and substantia nigra that correspond to signs of Parkinsonism. A more than four-fold increase of specific antibodies against JEV proved to be the cause of his disease. A total of 40 grams of IVIG containing anti-JEV antibodies was administered in two courses separated by one month. His symptoms ameliorated after each course. Although there were residual symptoms on discharge to rehabilitation, the treatment was thought to have saved the patient’s life .There are four subtypes of DENV and they are known for more severe disease courses upon secondary infection. In such cases, a frequently observed symptom is a pronounced thrombocytopenia leading to haemorrhage, most likely due to bone marrow infection and, in addition, possibly due to the clearance of thrombocytes through macrophages. More than 1,000 deaths occur worldwide annually due to dengue haemorrhagic fever and is associated with an increased level of platelet-associated IgG . A randoWNV is originally endemic in East-Africa and the Middle East. Beginning with a couple of cases in New York City, between the years 1999 and 2002 WNV has conquered the USA through a bird-, horse-, mosquito- and human-cycle. WNV disease, including neurological involvement, has a high mortality in the range of 5 to 14% ,34. EighAcute flaccid paralysis is a life-threatening complication of WNV infection. A 55-year-old man with a quickly developing muscle weakness, reaching his respiratory muscles on the third day so that the patient needed mechanical ventilation was suspected to have a Guillain-Barré syndrome. Dexamethasone and plasmapheresis were administered. When on the sixth day WNV was suspected, corticosteroids and plasmapheresis were discontinued, and anti-WNV hyper immune high dose IVIG at 0.4 grams per kilogram body weight each day for a seven day course was started instead. This treatment led to the rapid improvement of his muscle weakness, so that the patient could be discharged to inpatient rehabilitation after one month . Since tet al. (2011). The case report is unusual, because the infection was transmitted through a transplanted liver. When WNV was suspected in this case, serum was taken for analysis and the patient received 0.4 grams of anti-WNV hyper immune IVIG per kg body weight on the fourth and eighth day of his hospitalization and recovered fully. On the fifth day, a positive IgM, both in serum and CSF samples, were obtained before the start of IVIG, and the MRI scans pointed to WNV. Final proof came through a positive WNV-PCR from a retained blood sample of the liver donor who had not shown any WNV-typical symptoms at the time of his death through intracranial haemorrhage during a hypertensive crisis [There are additional cases of WNV encephalitis mostly successfully treated with anti-WNV hyper immune high dose IVIG -42, amone crisis .WNV with the highest number of case reports of arboviral encephalitis treated with high dose IVIG is special, because anti-WNV hyper immune IVIG preparations are available. This means that, besides the immunomodulatory effects of generic high dose IVIG which is dampening hyper inflammatory states without a noteworthy accompanying immune suppression, there is also a specific protective anti-WNV effect exerted by those immunoglobulin preparations in animal experiments .Togaviridae family of the Alpha virus genus. EEE produces a mortality rate of about 30% and is the most severe of the mosquito-borne encephalitides. Poor outcome has been correlated with ages over 40, rapid progression to coma, severe hyponatremia, and a CSF white blood cell count above 500 at symptom onset [EEE is caused by EEE virus belonging to the om onset . A 69-yeom onset .Togaviridae family of the Alpha virus genus. CHIKV typically causes a febrile disease in Africa and South-East Asia that is characterized by severe joint pain and can become haemorrhagic causing encephalitic symptoms in rare cases. The virus has been imported to Europe through international travel, and outbreaks have already occurred in Europe, e.g. in Northern Italy. Three cases reporting neurological symptoms were described by Chusri et al. (2011). The cases showed hyper intense signals in the MRI scan. Patient 1 was a 27-year-old woman who recovered on supportive measures. Patient 2 was an 85-year-old man who had not recovered during a follow-up one year later. Patient 3, a 44-year-old woman, who suffered a very severe form of late onset CHIKV encephalitis, was given a four day course of high dose IVIG and quickly recovered upon administration of the immunoglobulins and had recovered completely at a follow-up after six months. It was not reported whether the IVIG preparation contained anti-CHIKV antibodies [CHIKV is a mosquito-borne virus belonging to the tibodies .et al. (1999) on the successful treatment of ADEM with high dose IVIG. Although in most cases of ADEM the trigger remains obscure, a mumps virus infection was found to be the cause for post-infectious ADEM in the third one of the described cases [High dose IVIG has also been reported to play an advantageous role in treating encephalitis induced by other viruses. Two severe cases of influenza A virus-induced encephalitis responded favourably ,47. Furted cases .The diverse case reports discussed above suggest that early intervention with high dose IVIG may be a general treatment option for viral and post-infectious encephalitides. Although IVIG is generally well tolerated, it is not free from side effects. Adverse effects are mostly mild that include aseptic meningitis, headache, fever, nausea, diarrhoea, blood pressure changes and tachycardia. More severe adverse effects include renal failure, thromboembolic events and anaphylactic reactions that are related to IgA deficiency. The prior exclusion of an IgA deficiency and the slow administration of IVIG in a sufficiently large liquid volume helps to minimize the serious side effects .in vivo proof and is still under debate. Although enhancement was seen in cultured macrophages or monocytes in vitro[With the exception of secondary DENV infection, ADE of flavivirus infection does not have any clear systematic  in vitro, it was  in vitro. Passive in vitro-8. ADE o in vitro. This ma in vitro. Thus, c in vitro or after in vitro-8, these in vitro.Active anti-TBEV vaccination continues to be the mainstay of TBE prophylaxis . Even wiIf this is the case, it may be important to obtain immunoglobulins from blood donors vaccinated against TBE , in analADE: Antibody-dependent enhancement; ADEM: Acute disseminated encephalomyelitis; arbo: Arthropod-borne; CHIKV: Chikungunya virus; CNS: Central nervous system; CSF: Cerebrospinal fluid; DENV: Dengue virus; EEEV: Eastern Equine encephalitis virus; IVIG: Intravenous immunoglobulin; JEV: Japanese encephalitis virus; MRI: Magnetic resonance imaging; SCID: Severe combined immune deficiency; TBE: Tick-borne encephalitis; TBEV: Tick-borne encephalitis virus; WNV: West-Nile virus; YFV: Yellow fever virus.The authors declare that they have no competing interest.DR, GD and HHN: wrote the paper; HHN: concept of the paper. All authors read and approved the final manuscript.DR is Head of the Department of Virology of the Veterinary Research Institute and Research Scientist at the Institute of Parasitology, Academy of Sciences of the Czech Republic. GD is Head of the Department of Virology and Rickettsiology at the Bundeswehr Institute of Microbiology in Munich. HHN is Research Virologist and Senior Clinical Microbiologist in the Institute for Medical Microbiology and Hygiene at the University of Regensburg.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/306/prepub


In human eyes, ocular enlargement/growth reflects active extracellular matrix remodeling of the outer scleral shell. Micro-RNAs are small non-coding RNAs that regulate gene expression by base pairing with target sequences. They serve as nodes of signaling networks. We hypothesized that the sclera, like most tissues, expresses micro-RNAs, some of which modulate genes regulating ocular growth. In this study, the scleral micro-RNA expression profile of rapidly growing human fetal eyes was compared with that of stable adult donor eyes using high-throughput microarray and quantitative PCR analyses.Scleral samples from normal human fetal (24 wk) and normal adult donor eyes were obtained , and RNA extracted. Genome-wide micro-RNA profiling was performed using the Agilent micro-RNA microarray platform. Micro-RNA target predictions were obtained using Microcosm, TargetScan and PicTar algorithms. TaqMan® micro-RNA assays targeting micro-RNAs showing either highest significance, detection, or fold differences, and collagen specificity, were applied to scleral samples from posterior and peripheral ocular regions . Microarray data were analyzed using R, and quantitative PCR data with 2^-deltaCt methods.11). Specifically, fetal sclera showed increased expression of mir-214, let-7c, let-7e, mir-103, mir-107, and mir-98 . However, no significant regionally specific differences .i.e., posterior vs. peripheral sclera, were observed for either adult or fetal samples.Human sclera was found to express micro-RNAs, and comparison of microarray results for adult and fetal samples revealed many to be differentially expressed (p<0.01, min p= 6.5x10For the first time, micro-RNA expression has been catalogued in human sclera. Some micro-RNAs show age-related differential regulation, higher in the sclera of rapidly growing fetal eyes, consistent with a role in ocular growth regulation. Thus micro-RNAs represent potential targets for ocular growth manipulation, related to myopia and/or other disorders such as scleral ectasia. A signaling cascade that originates in the retina is believed to direct the scleral changes underlying ocular enlargement or growth . During Micro-RNAs are small non-coding single-stranded RNAs, which serve as key regulators of gene expression at the post-transcriptional level. This regulation is achieved through base pairing with the 3’ untranslated region (UTR) of their target mRNAs, resulting in cleaving and degradation of the mRNAs, either by virtue of perfect or nearly perfect pairing (as seen in plants), or by translational repression resulting from imperfect pairing  -6. MicroMicro-RNA transcriptomes have also already been described for some ocular tissues, including the retina, lens, and cornea . As a model for the latter, we used normal human fetal eyes, with human adult eyes with stable ocular dimensions serving as our reference model. We used microarray analyses to establish and compare the scleral micro-RNA profiles of these two models. Results for a subset of micro-RNAs identified as having the potential to regulate collagen were validated using quantitative PCR micro-RNA assays in follow-up experiments. Although our primary interest is in myopia, obtaining human donor tissue from young eyes with progressing myopia is difficult, if not impossible. Thus fetal eyes were selected as a substitute for the latter in this initial search for key micro-RNA regulators of scleral remodeling during accelerated ocular growth. During gestation, the rate of ocular axial elongation in humans is as high as 0.66 mm/week at 20 weeks and 0.32 mm/week at 30 weeks . A 24-weIn brief, we found that the human sclera expresses many micro-RNAs, some of which show age-related differential expression, consistent with a role in ocular growth regulation. A link between the scleral micro-RNAs confirmed to be up-regulated in fetal sclera and myopia, which also involves rapid ocular enlargement, is plausible but yet to be established.Normal human adult eyes (55 to 80 years of age) and normal fetal eyes (24 week gestation) were obtained from the North Carolina Eye Bank and Advanced BioResources , respectively. The study was approved by Duke University’s Institutional Review Board and adhered to the tenets of the Declaration of Helsinki guidelines.Comprehensive ocular histories were available for adult donor eyes and were reviewed to judge the suitability of the tissue. The histories included surgical history, syndromic connective tissue disorders, and form-deprivation myopia conditions , as well as refractive error status and in some cases, ocular axial length. Eyes with posterior segment ocular disease, including glaucoma and age-related macular degeneration, were excluded. Eyes of donors with significant systemic conditions such as cancer, requiring long-term drug therapies with possible effects on the eyes were also excluded. Systemic hypertension and diabetes were not among the exclusion criteria. For all adult donors, the cause of death was given as either heart or pulmonary disease, although their complete medical histories were not always available. Enucleated eyes were received and stored in RNAlater™ within 6 hours of death. In the adult donor group, all eyes were Caucasian and balanced for gender. Fetal donor eyes were collected from elective abortions; none were pre-term and thus not exposed to oxygen. All fetal eyes were harvested within minutes of death for storage in RNAlater™. Fetal samples were only balanced for gender since ethnic information was not available. Although only scleral samples were used in the study described here, retina, RPE-choroid complex and sclera samples were all collected. These tissues were separated by fine dissection from 5 mm trephine punches taken from the posterior pole region that included the macular region, where most of the scleral distension and related retinal thinning occurs in high myopia. An additional, more peripheral punch was collected at least 8 mm away from the fovea, the decision to collect this additional sample being driven by recent interest in the role of the peripheral retina in the regulation of refractive errors and ocular shape  20]. Sc. Sc20]. In processing scleral samples, they were first homogenized  and then total RNA extracted using a miRVANA™ isolation kit (Ambion Inc.), which is designed to preserve the small RNAs. The quality and quantity of RNA were determined using a Nanodrop™ (Thermo Scientific Inc.) and/or an Agilent Nanochip together with a Bioanalyzer (Agilent Inc.) ). Only samples with A260/A280 ratios of greater than 1.8 and RIN score greater than 7 were further processed.Profiling experiments used posterior and peripheral scleral samples from fetal and adult eyes , combined with Agilent Human micro-RNA microarrays  and high-performance SurePrint Technology™. The arrays were in an 8-plex format (8 arrays per slide), with 60-mer probes and 16-20 replicate features per micro-RNA. A total of 866 (Rel 12.0) or 1205 (Rel 16.0) human micro-RNAs were represented on the microarrays. Micro-RNA profiling experiments were processed at Asuragen Services , according to standard operating procedures in a GLP-compliant services laboratory. Briefly, total RNA was first dephosphorylated and then the pCp-Cy3 labeling molecule ligated to the 3’ end of the RNA molecules. The labeled RNA was purified using BioSpin6 . Hybridization, washing, staining, imaging, and signal extraction were performed according to Agilent-recommended procedures. http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/), TargetScan (http://www.targetscan.org/) and PicTar (http://pictar.mdc-berlin.de/) algorithms  and targeted micro-RNAs showing collagen (Col1A1) specificity  and either highest significance, detection, or fold differences in comparisons of equivalent fetal and adult samples in microarray profiling. Micro-RNA target predictions were obtained using Microcosm (http://www.r-project.org). Arrays within a specific experiment were normalized together according to the Variance Stabilization and Normalization (VSN) method described by Huber et al. [The signal processing implemented for the Agilent micro-RNA array was a multi-step process involving probe specific signal detection calls, background correction, and global normalization. For each probe, the contribution of signal due to background was estimated and removed by the Agilent Feature Extraction software as part of the data file output. Detection calls also made use of the Agilent Feature Extraction software. All other analyses were performed in R  to 0.05. A method described by Benjamini & Hochberg [k) with k being the largest value of i for which pi<(i/m)q in order to control the FDR at level q when the m distinct p-values pi are independent. Empirical Bayes methods (as implemented by the LIMMA R package), were used to shrink individual probe/micro-RNA variances towards a common value, also augmenting the degrees of freedom for the individual variance estimates [Principal Components Analysis (PCA) was performed after first normalizing and then mean-centering the data for each probe to check the variance in the dataset. Individual variances are not rescaled in PCA, so that genes with larger variance will have greater weight in determining the principal components than genes with lower variance. Mean expression values, standard deviations, and logHochberg  was usedstimates . Both thQuantitative PCR data were analyzed using the Comparative CT method, where the amount of target is normalized to endogenous reference/s, and 2^-deltaCt values and fold differences between groups calculated . The geo10 of p-values  and the x-axis shows the difference in the intensity values in the log 2 space between the two groups being compared. The data points are color-coded, with red markers signifying micro-RNAs with false discovery rate (FDR) corrected p-value of less than 0.05. A significant number of micro-RNAs showed significant age-related differences in expression, while far fewer micro-RNAs showed regional (ocular) differences in expression within each of the two age groups. Shown in The comparisons of equivalent adult and fetal samples . The relAnalysis of the variance in the data provides one way of examining differences between groups, as in the PCA plot shown in Cluster analysis of micro-RNA expression patterns aided in identifying sets of micro-RNAs that were differentially regulated in specific group-wise comparisons . From this analysis, it is clear that differences between adult and fetal groups represent the major differences overall. Clusters are depicted in That many scleral micro-RNAs show significant, age-related differential expression, as revealed in microarray experiments, was confirmed with quantitative PCR assays, which used a slightly expanded cohort . Our study represents the first comprehensive study of micro-RNA expression in the sclera. We not only established a catalogue of micro-RNAs for the human sclera, but in comparing scleral expression profiles of fetal and adult human eyes, we also identified a subset of micro-RNAs that could potentially be involved in ocular growth regulation. The findings of this study are discussed in the context of matrix remodeling and potential roles for micro-RNAs in abnormal ocular growth, such as myopia, which is our primary research interest.Our study revealed significant differences in the scleral micro-RNA expression profile of fetal eyes compared to that of adult eyes. Because fetal eyes are rapidly growing while the eyes of older adults are likely to be relatively stable in their overall dimensions, we rationalized that some of the identified differentially expressed micro-RNAs may be involved in modulating gene expression critical to ocular growth regulation. Furthermore, since the rate of ocular expansion primarily reflects scleral collagen regulation and so extracellular matrix remodeling, focusing our efforts on micro-RNAs known to be involved collagen gene regulation would seem most likely to capture those involved in ocular growth regulation.  To-date, a number of micro-RNA families have been implicated in collagen gene regulation in cardiovascular, liver, muscle, renal and eye diseases  -30, withOf the seven micro-RNAs selected for follow-up quantitative PCR analysis from the larger dataset generated from our microarray analyses , all but two of these micro-RNAs (hsa-let7b and hsa-let7c) showed significant up-regulation in fetal compared to adult sclera, irrespective of whether tissue samples were collected from the posterior pole or more peripherally. In the case of hsa-let7b and hsa-let7c, only posterior scleral samples showed significant differences in expression between fetal and adult eyes. Noteworthy perhaps is that the micro-RNA let-7 family has recently been implicated in collagen modulation in pancreatic cancer cells . Future in vitro study by Chen et al. [Relatively little is known about the functions in gene regulation of micro-RNAs , although one member of this group, mir-214, has been implicated in cell survival in cancer  and anotn et al. .As mentioned previously, working within the limitations of ocular donor tissues, we made every possible effort to control for variables unrelated to ocular growth that could confound our results. Nonetheless, we must also acknowledge three potentially important limitations of our studies: 1) the lack of systemic medical histories of some adult donors, 2) the use of differences in donor age as a surrogate for differences in ocular growth rates, and 3) potential sources of micro-RNAs in the sclera other than fibroblasts. In relation to the first point, we believe that undocumented systemic diseases are not likely to have biased our results on the basis that it is unlikely that systemic disease conditions were significantly over-represented in donors with incomplete records. Furthermore, any increase in the noise in our data level, a more likely consequence of undocumented systemic disease, would have reduced our ability to detect expression differences related to age alone. In relation to the second point, it is possible that the mechanisms underlying rapid, albeit normal ocular expansion during fetal development are not synonymous with those underlying the accelerated ocular expansion underlying myopia. It is also possible that additional factors, apart from the high rate of ocular growth of fetal eyes, are reflected in their scleral micro-RNA profiles. Nonetheless, the identified subset of scleral micro-RNAs, linked to both rapid normal ocular growth and collagen gene regulation, would seem a reasonable starting point for investigations aiming to identify scleral micro-RNAs involved in “myopic” growth. That the study yielded a catalogue of micro-RNAs for fetal and adult human sclera also represents a valuable resource beyond myopia, given the increasing interest in this tissue in relation to glaucoma, among other ocular diseases ,36. AddrIn summary, this is the first comprehensive study of micro-RNA expression in human sclera. The sclera was found to express micro-RNAs, some of which show age-related differential regulation, higher in rapidly growing fetal eyes, consistent with a potential role in ocular growth regulation. A role for these scleral micro-RNAs in the development of myopia, which also involves accelerated eye growth, is thus plausible. The current findings provide a platform for new lines of mechanistic/pathway-related investigations directed at understanding the role of micro-RNAs in scleral remodeling. Understanding the role of micro-RNAs in scleral mechanisms underlying myopia may open up new anti-myopia treatment strategies. Table S1micro-RNA predictions for Col1a1.(XLS)Click here for additional data file.Table S2Summarized values.(XLSX)Click here for additional data file.Table S3Gene expression differences.(XLSX)Click here for additional data file.Table S4Clusters.(XLSX)Click here for additional data file.Figure S1Pearson correlation.(TIF)Click here for additional data file.


Adults born preterm at very low birth weight (VLBW ≤ 1500g) have increased risk factors for cardiovascular diseases including high blood pressure and impaired glucose regulation. Non-optimal lipoprotein profile is generally also likely to affect the increased cardiovascular risk, but lipoprotein subclass level data on adults born at VLBW are sparse.We studied 162 subjects born at VLBW and 169 term-born controls, aged 19 to 27 years. Total lipid, triglyceride and cholesterol concentrations of 14 lipoprotein subclasses were determined by proton nuclear magnetic resonance spectroscopy in the fasting state and in 2-hour serum samples from an oral glucose tolerance test.In comparison to controls, VLBW subjects had significantly higher fasting concentration of triglycerides in chylomicrons and largest very-low-density lipoprotein particles , and of triglycerides in small high-density lipoprotein particles . The seemingly important role of triglycerides was further supported by principal component analysis in which the first component was characterized by multiple lipoprotein triglyceride measures.Young adults born at VLBW and their peers born at term had triglyceride-related differences in both VLDL and HDL subclasses. These differences suggest that the increased risk factors for cardiovascular diseases among the VLBW individuals in adulthood may partly relate to impaired triglyceride metabolism. Adult-onset cardiovascular diseases have their origins partly in intrauterine and early postnatal life . As younHere we investigated lipoprotein metabolism at subclass level in VLBW and control subjects from the Helsinki Study of Very Low Birth Weight Adults by proton nuclear magnetic resonance (NMR) spectroscopy.The participants are from a longitudinal follow-up cohort of subjects born preterm at VLBW between 1978 and 1985 and discharged alive form the neonatal intensive care unit of Children's Hospital, Helsinki University Central Hospital, Finland. Of the original 335 survivors, 255 VLBW subjects lived in the greater Helsinki area at the time of follow-up and were invited to a clinical study together with a sex-, age-, and birth hospital-matched comparison group of 314 term-born subjects who were not small for gestational age (birth weight more than −2 SD) ; 166 VLBAfter an overnight fast of at least 8 hours, the clinical examination included weight and height measurement, and a 2-hour 75 g OGTT  .We then performed a We observed that, in comparison to controls, adults born at VLBW have higher concentrations of triglycerides both in the largest VLDL particles and in small HDL particles. These potentially atherogenic characteristics of the lipid profile  may contIn the general population a weak negative correlation has been reported between birth weight and serum total cholesterol . SimilarA strength of this study is the measurement of extensive lipoprotein subclass data at both in the fasting state and 2-hours after an oral glucose challenge. We were also able to adjust for most potential confounding factors, such as body mass index, smoking, and parental education. These factors did not explain the observed difference between VLBW and control subjects. A common challenge, as also here, in the studies of VLBW individuals is the relatively small number of study participants available. Combined with the rather high number of lipoprotein measures analysed, this created a statistically difficult situation; however, the trends for biologically related variables supported the findings as well as reduction of the outcome variables using principal component analysis. The remaining skewedness in the log-transformed variables may, in general, cause false negative findings. During the primary analysis, we further ‘Box-Cox transformed’ the variables when it was suspected logarithmic transformation did not adequately remove the skewedness. After this further transformation the difference estimates did not increase, but decreased, approximately by 50% at maximum. Thus, these two methods, together with the principal component approach, support VLBW adults exhibit a difference in the extremely large VLBW triglycerides and total lipids.In conclusion, we found atherogenic characteristics in the lipoprotein profile of young adults born preterm at VLBW. The differences between the VLBW and the term-born individuals were not detectable with conventional lipid measurements. These findings, though subtle, may indicate that impaired triglyceride metabolism partly contributes to the increased risk for cardiovascular disease among adults born severely preterm.ApoA1: Apolipoprotein A-I; ApoB: Apolipoprotein B; C: Total cholesterol; D: Diameter; HDL: High-density lipoprotein; IDL: Intermediate-density lipoprotein; LDL: Low-density lipoprotein; L: Total lipids; NMR: Nuclear magnetic resonance; OGTT: Oral glucose tolerance test; PC: Principal component; SGA: Small for gestational age; TG: Triglycerides; VLBW: Very low birth weight; VLDL: Very-low-density lipoprotein.The authors declare that they have no competing interests.KW took part in carrying out the statistical analyses, participated in planning of the study, and drafted the manuscript. PH took part in carrying out the statistical analyses, and participated in writing the manuscript. PH, EK, SA, A-LJ, and JGE participated in the design of the clinical study. PH, EK, SA, and A-LJ collected the study participants and PH, EK, SA, and JGE performed the clinical studies. PS, AK, and MA-K designed and performed the NMR analyses. MA-K participated in the statistical interpretations as well as in writing the manuscript. All authors read and approved the final manuscript.


Rana catesbeiana from tadpoles to froglets using ultraperformance liquid chromatography coupled to a mass spectrometer. Total and aqueous metabolite extracts were obtained from each serum sample to select for nonpolar and polar metabolites, respectively, and selected metabolites were validated by running authentic compounds.After completion of embryogenesis, many organisms experience an additional obligatory developmental transition to attain a substantially different juvenile or adult form. During anuran metamorphosis, the aquatic tadpole undergoes drastic morphological changes and remodelling of tissues and organs to become a froglet. Thyroid hormones are required to initiate the process, but the mechanism whereby the many requisite changes are coordinated between organs and tissues is poorly understood. Metabolites are often highly conserved biomolecules between species and are the closest reflection of phenotype. Due to the extensive distribution of blood throughout the organism, examination of the metabolites contained therein provides a system-wide overview of the coordinated changes experienced during metamorphosis. We performed an untargeted metabolomic analysis on serum samples from naturally-metamorphosing padj < 0.001) between metamorphic stages. We observed extensive remodelling of five core metabolic pathways: arginine and purine/pyrimidine, cysteine/methionine, sphingolipid, and eicosanoid metabolism and the urea cycle, and found evidence for a major role for lipids during this postembryonic process. Metabolites traditionally linked to human disease states were found to have biological linkages to the system-wide changes occuring during the events leading up to overt morphological change.The majority of the detected metabolites (74%) showed statistically significant abundance changes (To our knowledge, this is the first wide-scale metabolomic study of vertebrate metamorphosis identifying fundamental pathways involved in the coordination of this important developmental process and paves the way for metabolomic studies on other metamorphic systems including fish and insects. After embryogenesis, many organisms experience obligatory developmental transitions to successfully move from one ecological niche to another. One such transition is through metamorphosis in which an immature larva transforms into a juvenile or adult scarcely resembling the initial form. Classic examples occur in vertebrates and invertebrates alike, and often require the involvement of hormone signaling systems. However, a fundamental question in biology remains in understanding how a fully-differentiated organism coordinates the many tissue- and organ-system changes during the metamorphic process ,2.Frog tadpoles undergo significant morphological changes, resulting in the development of limbs, resorption of the tail, and a shift from gill to lungs in respiratory organs used, hence a shift from purely aquatic to a semi-terrestrial lifestyle and a change in diet. This extensive process involves apoptosis, cell proliferation, and reprogramming and highlights the complexity, tight regulation, and interconnection of biological networks and pathways.Despite such complexity, anuran metamorphosis is initiated solely by thyroid hormones (THs) and this important postembryonic developmental period can be divided into three specific stages: premetamorphosis, prometamorphosis and metamorphic climax, characterized in part by TH status . PremetaMetabolomics is the comprehensive analysis of the whole metabolome (metabolite profiles) under a given set of conditions  and is aRana catesbeiana (North American bullfrogs) at different postembryonic developmental stages: from tadpoles to froglets. Serum was the tissue of choice to provide an overall view of the dynamic changes experienced by the frog tadpole and enable the identification of metabolites involved in the coordination of metamorphic processes throughout the tadpole. R. catesbeiana were used in the present study because of their large size enabling the analysis of serum from individual animals, their world-wide distribution and availability, and their genetic diversity and life history resemble that of humans more closely than other laboratory frog species + and [M + Na]+ were selected as possible adducts while [M-H]- was chosen as a possibility for the ESI- generated data (Tot- and Aqu-). The allowable mass error was set to 0.02 Da, and KEGG/HMDB/LIPID MAPS [Rana catesbeiana was not available as a choice of organism, Homo sapiens was chosen due to the completeness of the database and the similarities in genetic diversity and metabolomic systems. In the optional pathway analysis field, 90 different KEGG pathway IDs were pasted in order to obtain a comprehensive coverage of the possible locations of the metabolites in metabolic pathways. These pathways included the citric acid cycle, fatty acid synthesis, steroid hormone biosynthesis, amino acid metabolism and degradation, etc. When there were multiple hits for the possible IDs of one metabolite, the most likely ID was inferred based on the chemical profile of the metabolite . We focused only on those masses with only one possible ID or where all but one ID had been eliminated by chemical profile evaluation. Using the KEGG pathway maps in which the locations of query metabolites were highlighted, we connected and constructed metabolic pathway maps. For each metabolite, the integration values at the indicated TK stage ranges were compared to the values at the premetamorphic stage as a control, using the nonparametric multiple comparison procedure for unbalanced one-way factorial design proposed by Gao et al.[p-values were FDR-corrected, and a significance level of 0.05 was used.The metabolite features whose abundance profiles showed significant heteroscedacity at a certain TK stage or significant abundance changes were searched against the MassTRIX ver. 3 webserver  (http://PID MAPS -88 withoao et al.. The p-vAuthentic compounds for the selected metabolites were obtained as shown in Table C1P: Ceramide 1-phosphate; CB1: Cannabinoid type 1; ESI: Electrospray ionization; FDR: False discovery rate; HETE: Hydroxyeicosatetraenoic acid; HHH: Hyperammonemia, hyperornithinemia, homocitrullinuria; HPETE: Hydroperoxyeicosatetraenoic acid; ID: Identities; L-DOPA: L-3,4-dihydroxyphenylalanine; LT: Leukotriene; MS: Mass spectrometry; NADA: N-arachidonoyldopamine; NMDA: N-methyl-D-aspartate; NO: Nitric oxide; PCA: Principal components analysis; PG: Prostaglandin; PRL: Prolactin; PS: Phosphatidyl serine; Q-TOF: Quadrupole time-of-flight; R.: Rana; RT: Retention time; S1P: Sphingosine 1-phosphate; SAH: S-adenosylhomocysteine; SAM: S-adenosylmethionine; TH: Thyroid hormone; TG: Triglyceride; TK: Taylor Kollros; TR: Thyroid hormone receptor; TRPV1: Transient receptor potential vanilloid type 1; UPLC: Ultra-performance liquid chromatography.The authors declare that they have no competing interests.TI isolated the serum, performed the mass spectrometry, data, and statistical analyses, and drafted the manuscript. JH performed mass spectrometry and data analysis. CB participated in the design of the study and provided reagents/materials/analysis tools. ML participated in the experimental design and statistical analysis. CH conceived of the study, and participated in its design and coordination and helped draft the manuscript. All authors read and approved the final manuscript.