Attachment insecurity relates to the onset and course of chronic pain via dysfunctional reactions to pain. However, few studies have investigated the proportion of insecure attachment styles in different pain conditions, and results regarding associations between attachment, pain severity, and disability in chronic pain are inconsistent. This study aims to clarify the relationships between insecure attachment and occurrence or severity of chronic pain with and without clearly defined organic cause. To detect potential differences in the importance of global and romantic attachment representations, we included both concepts in our study.85 patients with medically unexplained musculoskeletal pain (UMP) and 89 patients with joint pain from osteoarthritis (OA) completed self-report measures of global and romantic attachment, pain intensity, physical functioning, and depression.Patients reporting global insecure attachment representations were more likely to suffer from medically unexplained musculoskeletal pain (OR 3.4), compared to securely attached patients. Romantic attachment did not differ between pain conditions. Pain intensity was associated with romantic attachment anxiety, and this relationship was more pronounced in the OA group compared to the UMP group. Both global and romantic attachment anxiety predicted depression, accounting for 15% and 17% of the variance, respectively. Disability was independent from attachment patterns.Our results indicate that global insecure attachment is associated with the experience of medically unexplained musculoskeletal pain, but not with osteoarthritis. In contrast, insecure attachment patterns seem to be linked to pain intensity and pain-related depression in unexplained musculoskeletal pain and in osteoarthritis. These findings suggest that relationship-informed focused treatment strategies may alleviate pain severity and psychological distress in chronic pain independent of underlying pathology. The experience of chronic pain is multifactorial, and neither merely biologically determined nor a purely psychogenic phenomenon. Current models conceptualize chronic pain as resulting from complex interactions between biological and psychosocial factors . Psychosattachment from other, especially organic factors by identifying attachment patterns and associations between attachment and symptom severity in two different groups of patients with musculoskeletal pain: One with a clearly defined organic cause  and one without .Chronic pain may or may not have an underlying organic cause in terms of a medically identifiable pathology. However, in both cases, physiological and psychological changes will occur as a consequence . These cAdult attachment theory provides a developmental framework for interindividual differences in cognitive, emotional, and behavioral responses to pain. Insecure adult attachment patterns are considered a risk factor for the development of chronic pain, as well as poor prognosis in the face of chronic pain . AccordiInternal working models have been conceptualized in recent studies as hierarchically organized neural network structures, running from memories of particular relationship partners, through representations of different relationship domains , to central tendencies, which form a context-sensitive, generic attachment working model . BecauseInsecure attachment is a widely acknowledged risk factor for not only psychopathology, but also adverse health behavior and outcome in a variety of health conditions –19. ReceHowever, results on direct associations between attachment patterns and pain intensity and disability among patients with chronic pain are mixed : some auIn sum, insecure attachment is associated with dysfunctional reactions to acute pain and psychological distress in chronic pain. Inconsistent findings with regard to pain intensity and disability can be attributed to undetected differences between specific chronic pain conditions, or between differences in assessment of general vs. romantic attachment patterns. Based on the evidence linking attachment theory with chronic pain, Meredith  presenteThe present study seeks to contribute to the understanding of the potentially differential impact of attachment as a dispositional vulnerability factor for the occurrence of chronic pain conditions, or as a factor affecting their progression and severity. To address these questions, we systematically compared two different pain subgroups, one with , and one without  a clearly defined organic cause. To enhance measurement precision, improve comparability to other major studies, and detect potential differences in the importance of global vs. romantic attachment patterns, we assessed both concepts in our study.Insecure attachment partially accounts for interindividual differences in the responses to acute pain, which are crucial for the transition to chronic pain . DysfuncPatients with insecure attachment patterns adapt to pain in more dysfunctional ways than securely attached patients . EmpiricTwo clinical samples were investigated prior to treatment in a cross-sectional design. Patients of both samples suffered from chronic musculoskeletal pain for a continuous period of at least six months. They presented themselves to the Department of Orthopedic Surgery of the University of Heidelberg between September 2008 and January 2010. At the time of admission, comprehensive medical and psychological diagnostic procedures were administered. To check whether pain intensity, pain location, or pain spreading was fully explained by specific somatic pathology, the results of a clinical examination were compared to diagnostic imaging by an orthopedic specialist. Furthermore, the Structured Diagnostic Interview for DSM-IV Axis I Disorders (SCID-I) was administered by a trained psychologist to detect mental disorders. Patients were included in the UMP group, if 1) they suffered from CWP , back paWe calculated the necessary sample size for the entire sample (UMP and OA) with the G*Power Analysis software program for logistic regression analysis, and by reference to our primary hypothesis with regard to the secure-insecure RQ-2 attachment style categories. We expected a moderate effect of OR 3.0 for the association between insecure attachment with UMP . The results indicated that 172 participants were required. Of the 243 patients eligible , 191 met the inclusion criteria . Of these, 174 (91%) consented to participate in the study and completed the baseline questionnaires  . Data weThe study procedures were approved by the Institutional Review Board of the Medical Faculty, University of Heidelberg (S-220/2008), and complied with the ethical standards established in the Declaration of Helsinki . DecisioSubjects were asked to report their age, gender, educational level, relationship and occupational status.The Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I) is a semi-structured interview for assessing DSM-IV Axis I diagnoses. It is considered diagnostic gold standard in psychiatric assessment, and has shown high reliability and superior validity when conducted by trained health professionals .As the human attachment system comprises representations of global models of the self and others in interaction and specific relationship experiences that are not perfectly correlated , we chosThe Relationships Questionnaire (RQ-2) is a brief self-report inventory that evaluates global attachment –11 accorTo assess romantic attachment, we used the German version  of the wSubjective pain intensity over the preceding week was measured on a visual analogue scale (VAS) ranging from 0 (no pain) to 100 (worst imaginable pain). Each patient indicated a position on the VAS in response to the question “How severe was your pain on average in the last seven days?” The VAS is a valid and reliable instrument for measuring the intensity of pain .Functional impairment was measured using the Hannover Dorsal Function Questionnaire (FFBH-R-R) , a questPsychological distress was measured by means of the depression subscale of the German version of the Hospital Anxiety and Depression Scale (HADS-D) , comprisDescriptive statistics, such as measures of central tendency and of dispersion, were used for basic statistical analysis. Depending on data type, parametric t-tests, ANOVA, Pearson’s chi-square and Fisher’s exact test, respectively, were used for basic group comparisons between UMP and OA. Correlations between the RQ and ECR-R dimensions were assessed by means of Pearson’s r. To test the first hypothesis, odds ratios and confidence intervals from logistic regression models were used to quantify attachment styles in both groups. Separate linear regression models were constructed to examine the associations of global or romantic attachment with pain intensity, disability, and depression. In the logistic and linear regression models, independent variables were included in a forced-entry fashion. Before applying the regression analysis, the variables were tested for normal distribution via the Kolmogorov-Smirnov test, and graphical interpretation. All tests for significance were two-sided; p values below. 05 were considered statistically significant. Statistical analysis was performed using IBM SPSS Statistics 21.Missing data resulted in decreased N values for regression analyses. Post hoc power calculation for logistic regression analysis with the given sample size of 148 patients with complete data for the examination of our first research question indicated a power of 80% with regard to the logistic regression analysis based on the secure-insecure categories, and of a power > 80 with regard to the analysis based on the four attachment style categories.Descriptive statistics for demographic and clinical characteristics are shown in The number and frequency of mental disorders diagnosed by means of the SCID-I are shown in 2 (1) = 11,24, p = .002): about one-third of UMP patients were classified as securely attached (38.8%), while this was true for two-thirds (64.2%) of OA patients. Preoccupied and fearful attachment styles were characterized by a negative model of self. The increased proportion of these categories is also reflected in lower dimensional RQ model of self scores in UMP compared to OA. In contrast, levels of the romantic ECR-R dimensions did not differ between the groups  = 0.433, p <. 001 for RQ model of other and ECR-R attachment avoidance.The frequencies of global RQ attachment style categories are presented in e groups . RQ scormodel of self and RQ model of other dimensions as independent variables. Age, educational level, relationship status, and RQ model of self  were significant predictors . Global attachment (RQ model of self /RQ model of other) and romantic attachment (ECR-R attachment anxiety/ECR-R attachment avoidance) were entered in the third block, respectively. In the fourth block, the interaction between pain condition and global attachment, or romantic attachment, were entered. Standardized beta regression coefficients for the predictors (except for the control variables) and R2 values of the regression models are shown in Tables To test our hypothesis that insecurely attached patients in both pain conditions will report higher levels symptom severity , hierarchical multiple regression analyses were conducted separately for each attachment measure . We controlled for gender, age, education, and relationship status. Moreover, the clinical characteristics not predicted by the model were entered as covariates due to the well-known interaction between depression, pain perception, and disability in chronic pain patients –2. In thmodel of self  were significantly related to depressive symptoms . There was also a significant interaction between attachment anxiety and pain condition for pain intensity  s. . In otheThe present study sought to inform our understanding of insecure attachment as a dispositional vulnerability for the occurrence and severity of chronic pain. We investigated two well-defined, adequately large samples of patients with and without a clear somatic pathology. This is the first study to use two measures assessing different attachment domains in chronic pain. The investigation of pain syndromes with different etiologies, and the combination of attachment measures allowed us to detect differences between pain conditions and types of attachment representations, helping to clarify inconsistencies in previous research.We hypothesized that insecure attachment patterns are more frequently associated with medically unexplained musculoskeletal pain, compared to pain from severe osteoarthritis. This was confirmed for global attachment representations, but not for romantic attachment domains. There was a higher prevalence of global insecure attachment styles in UMP (nearly 2/3), whereas the proportion of 36% global insecure attachment styles in osteoarthritis matches the proportion found in a large nationally representative sample of adults from the United States . Age, edInterestingly, romantic attachment insecurity was not directly related to medically unexplained pain. Romantic attachment anxiety and romantic attachment avoidance did not differ between pain conditions and were similar to those of nonclinical samples . These dWe also expected attachment insecurity to be associated with pain intensity, disability, and depression in both pain conditions. Our predictions were confirmed with regard to pain and depression, but not disability. Pain intensity and functional impairment were independent of global attachment, across secure/insecure categories, and model of self/other dimensions. These results support earlier findings –27, suggRegarding depression, consistent associations were found in both groups and measures. Lower global attachment model of self-scores were associated with higher depression in both pain conditions. Similar results were obtained using the romantic attachment measure. Patients with higher romantic attachment anxiety were likely to report higher psychological distress in terms of depression. The presence of a somatoform disorder as a mental health condition in the UMP group yielded no effects of attachment by group interactions on depression. While associations between adult attachment and depression have been documented in other samples –52, our Our results must be regarded as preliminary due to some methodological limitations. First, owing to the cross-sectional nature of our study, it remains unclear whether the experience of UMP influences the activation of fearful or preoccupied attachment , or whetOverall, the results supported our prediction that insecure attachment is more prevalent in medically unexplained pain compared to pain with a clear organic cause, but that poorer outcome in chronic pain relates to insecure attachment independently of organic pathology. As described above, our study revealed substantial differences in the relationships of global vs. romantic attachment patterns with the occurrence and outcome of chronic pain. First, medically unexplained pain was associated with global attachment insecurity, but not with romantic attachment insecurity. Second, pain intensity was linked to romantic attachment anxiety, but not to global attachment insecurity. These differences between the levels of attachment representation in chronic pain have implications for the development of attachment-informed strategies in prevention and treatment. Therefore, our results underline the need to integrate generic and relation-specific attachment representations in research approaches to increase the understanding of their differential importance in the context of chronic pain.Our results tentatively suggest the importance of incorporating attachment-informed interventions into the prevention and rehabilitation of chronic pain. The finding that general patterns of insecure attachment are associated with UMP requires early intervention strategies that consider the specific interactional needs that are linked to these attachment styles. For example, patients with a preoccupied attachment style may be impaired in their ability to benefit from self-management-focused treatment strategies, and may need more reassuring, supporting approaches to gradually foster their self-efficacy and independence, whereas patients with a fearful attachment style may need encouragement towards self-disclosure and appropriate help-seeking behavior. Our findings argue against a direct impact of global attachment patterns on the severity of pain and disability. However, they confirm earlier observed associations of insecure romantic attachment and pain intensity, suggesting the importance of involving close others in treatments to reduce the experience of pain with and without clearly identifiable pathology. As global and romantic attachment insecurity were linked to the severity of depressive symptoms in both unexplained musculoskeletal pain and osteoarthritis, rehabilitation programs for chronic pain syndromes should consider not only depression but also different forms of attachment insecurity by implementing relationship-focused approaches.S1 DatasetThis data file contains raw data from questionnaires measuring attachment, pain, disability and depression in two different pain conditions. All regression analyses are based on these data. The file includes pain condition (pain_condition), age (Age), sex (Sex), living in partnership (Partnerschaft_kat), college entrance (Education), employment (Employment), sick leave (EM_Rente_jn), pain intensity last seven days (Schmerz_1_t1), raw data from HADS, FFbH, ECR-R, RQ, global attachment dimensions , global attachment styles (RQ_4Bindungsstile).(SAV)Click here for additional data file.


Developmental immunotoxicity (DIT) is a term given to encompass the environmentally induced disruption of normal immune development resulting in adverse outcomes. A myriad of chemical, physical, and psychological factors can all contribute to DIT. As a core component of the developmental origins of adult disease, DIT is interlinked with three important concepts surrounding health risks across a lifetime: (1) the Barker Hypothesis, which connects prenatal development to later-life diseases, (2) the hygiene hypothesis, which connects newborns and infants to risk of later-life diseases and, (3) fetal programming and epigenetic alterations, which may exert effects both in later life and across future generations. This review of DIT considers: (1) the history and context of DIT research, (2) the fundamental features of DIT, (3) the emerging role of DIT in risk of noncommunicable diseases (NCDs) and (4) the range of risk factors that have been investigated through human research. The emphasis on the human DIT-related literature is significant since most prior reviews of DIT have largely focused on animal research and considerations of specific categories of risk factors . Risk factors considered in this review include air pollution, aluminum, antibiotics, arsenic, bisphenol A, ethanol, lead (Pb), maternal smoking and environmental tobacco smoke, paracetamol (acetaminophen), pesticides, polychlorinated biphenyls, and polyfluorinated compounds. Early-life environmental insults affecting the developing immune system can have significant health ramifications not only for the exposed offspring but also potentially extending to additional generations. Developmental immunotoxicity (DIT) appears to play a significant role in the current global epidemic of non-communicable diseases (NCDs) , 2. ThisImmunotoxicology, the study of the adverse impact of environmental conditions  on the immune system, began to gain recognition during the 1970s and early 1980s  with theThe examination of adverse insults to the developing immune system, a subsection of immunotoxicology known as developmental immunotoxicity (DIT), was among the first research initiatives within immunotoxicology. As early as the 1970s, animal studies revealed the persistent nature of immune problems resulting from early-life insult. Studies involving drugs , 16, heaDIT did not achieve a priority position for research and safety evaluation within immunotoxicology until approximately the late 1990s–early 2000s. Among the important events were a workshop on childhood health risks coordinated by the March of Dimes and EPA –28, the is directly linked with immune dysfunction and increased risk of NCDs,stems from critical developmental windows of immune vulnerability restricted to the young,can happen at lower exposure levels than usually produce adult-exposure immunotoxicity,often involves a broader spectrum of adverse immune outcome versus adult-exposure immunotoxicity,usually produces more persistent effects than those following adult exposure,can lead to latent dysfunction that may be masked until it is triggered by a later-life event,often manifests as immune dysfunctional imbalances ,may produce different sex-based outcomes,is not routinely assessed in most required safety testing protocols to date,can occur via several different biological pathways .The DIT literature is sufficiently extensive to permit fundamental characterizations. This information is derived from [DITved from , 33–40.The impetus for a greater focus on DIT was aided by the findings of Barker and colleagues that maternal undernutrition during prenatal development could increase the risk of cardiovascular disease (CVD) in the offspring –43. ThisAs the net was cast beyond just maternal-fetal nutritional status to include a wide array of environmental conditions and factors, the concept of developmental origins of health and disease (DOHaD) emerged , 48 to cOne of the hallmarks of the developing immune system is that it exhibits an increased sensitivity for most environmentally induced toxicity compared with the fully matured immune system of the adult. Additionally, DIT often occurs at exposure doses that are below those producing other developmental effects –55. LuebThis increased risk of developmental immune insult compared with that of the adult has been seen across broad categories of drugs and chemicals as well as among different dietary and physical/psychological factors , 29, 37.The identification and consideration of both systemic and tissue-oriented developmental vulnerabilities for the immune system have undergone progressive evolution since the original series of immune “critical windows” emerged from a national workshop , 28. As Each immune developmental window has its own unique vulnerabilities that are best detected via age-relevant safety screening . For exaApplication of the critical windows concept for enhanced immune-associated disease prevention has been explored by Jenmalm and Duchén . These aOne of the outcomes of the recent human studies on DIT and fetal programming of immune-based disease is an increasing realization that these processes are major contributors to the ongoing epidemic of noncommunicable diseases (NCDs) (most of which are chronic diseases). NCDs are the major cause of death globally and include cardiovascular disease as well as cancer . What haOne of the impediments to recognizing this DIT-NCD linkage is that historic examination of immunotoxicity often focused on changes in primary and secondary immune organs. However, the majority of immune cells actually reside outside of these organs in mucosal and other tissues such as the gut, brain, skin, liver, endocrine, reproductive, urogenital, and cardiovascular systems. It is these tissues that are most often involved with NCDs, and the status of these resident immune cells is often at issue relative to tissue pathology. A shift in focus in immune evaluation to consider the impact of DIT and later-life status of cells such as skin dendritic cells, microglia, Kupffer cells, and immune cells of the BALT and GALT should provide a clearer picture of the cause-effect relationship between DIT and certain NCDs .  Table The significance of the prevention of DIT as a strategy to reduce the prevalence of NCDs has been strengthened with the awareness that NCDs exist as tightly intertwined patterns of comorbid risks. This paradigm of tightly interlinked chronic diseases and conditions was described in a series of papers illustrating the health risk trajectories that exist when children are diagnosed with a number of different immune-/inflammatory-driven conditions: asthma, recurrent infections, schizophrenia, autoimmune thyroiditis, celiac disease, inflammatory disease, and psoriasis , 67, 68.As an example, one interconnected pattern of comorbidity exists among a triad of autoimmune conditions: type 1 diabetes, celiac disease, and autoimmune thyroiditis. Children diagnosed with type 1 diabetes have a predictably greater risk for developing celiac disease and/or autoimmune thyroiditis , 71. WhiThe ramification of these comorbid disease interconnections is that there is increased value in avoiding fetal programming that results in childhood-onset, immune dysfunction-based NCDs. These implications led four immunotoxicologists to call for required DIT testing of chemicals and drugs as a step to better protect children from the risk of NCDs .Most prior reviews of DIT have focused largely on animal research. This section examines the wide range of risk factors for DIT that has been evaluated among human populations. Evidence supporting the occurrence of DIT among human populations has been obtained from both exposed populations as well as via epidemiological studies. The risk factors are presented alphabetically rather than being grouped into different categories .In many of these studies antibody titers against either a common virus or childhood vaccinations have been used as a biomarker of DIT. While limited as an overall immune measure, there are significant benefits to this approach: (1) serum antibody levels are easily determined, (2) a majority of children will have been vaccinated according to a predictable and standard schedule, and (3) the microbial infection or vaccine challenge of the child's immune system will enable a detection of potential dysfunction in an actively responding immune system and, based on animal data, these are among the most sensitive parameters for measuring DIT. Other studies reach beyond vaccination data to examine associations between exposure/environmental conditions and immune-based chronic diseases during childhood. Among the most commonly used are asthma, allergic rhinitis, atopic dermatitis, type 1 diabetes, celiac disease, and inflammatory bowel disease. Only a portion of these disease-association studies has overt human immune function associated with them. For the remainder, there has been a tendency to rely more on linking DIT immune function animal data with information on human immune disease-associations.Ambient air pollution including specific components  has been implicated in respiratory and cardiovascular diseases via improperly controlled inflammation. Nadeau et al.  examinedKerkhof et al.  found evγ and natural killer cells with evidence of elevated systemic inflammation (elevated C-reactive protein and prostaglandin E metabolites).Calderón-Garcidueñas et al.  comparedIndoor air pollution, beyond that of environmental tobacco smoke, which is discussed in a later section, has also been associated with human DIT. Herberth et al.  studied Aluminum exposure during prenatal and childhood development can occur via a variety of routes including via food, certain drugs , drinking water, and air  includinThere is evidence to suggest that febrile responses in children following alum-containing vaccination may represent an inflammation-driven hyperresponse that occurs in a subset of children, possibly those possessing certain cytokine gene alleles . A propoAntibiotic use in early life has been associated with an elevated risk of immune-based diseases such as childhood asthma. Raciborski et al.  found thExtensive antibiotic use is of particular concern when viewed in the context of the hygiene hypothesis or the recently-described “Completed Self” model   see Fig. Under tArsenic is found in both inorganic and organic forms. Most of the environmental health concerns have focused on the inorganic forms of arsenic  with exposure occurring primarily via ingestion of contaminated food and water and secondarily via inhalation. Some forms of arsenic  have been used in the treatment of leukemias. The topic of arsenic-induced immunotoxicity was recently reviewed by Dangleben et al. . These aStudies of exposed human populations also suggest that arsenic is a major concern for DIT, and several studies have examined children in Mexico and Bangladesh among highly exposed populations. Soto-Peña et al.,  found thThis suggests that, at physiologically-relevant exposures, arsenic-induced DIT can manifest almost immediately during fetal development. This is consistent with the findings of arsenic-exposed children in Mexico where increased arsenic levels were associated with increased basal nitric oxide production by monocytes and increased superoxide anion produced by activated monocytes . Taken tBisphenol A (BPA) is used in a variety of food and beverage containers. Most human chemical exposure occurs via food and beverages although exposure via air, dust, and water is also possible. Sources of BPA include food storage containers, water bottles, baby bottles, and polycarbonate tableware. BPA has received significant immune system evaluation in recent years although the majority of studies, to date, have been performed in rodents.Rogers et al.  recentlyHuman studies for BPA and DIT are comparatively limited to date. In a National Health and Nutrition Examination Survey (NHANES) study, children and teens less than 18 years of age exhibited an inverse correlation of BPA exposure (urinary levels of BPA) with antibody levels against cytomegalovirus . Kim et Cesarean section (CS) can be a medical necessity in some circumstances. However, the increased prevalence of elective CS ) has created a public health concern . CS has α, and IL-8) were significantly elevated with VD versus CS. The investigators suggested that increased sterile inflammation during labor and VD delivery may play a critical role in normal parturition and facilitate subsequent maturational processes  in the newborn [In the first category, birth delivery mode can significantly affect the microbiota and the subsequent immune-microbiome interactions. In a Canadian study, Azad et al.  found th newborn .α compared with CS delivered babies. Taken together, these studies suggest that neonatal immune profiles, including early inflammatory interactions, are locked into a less mature, more-fetal-like state following CS versus VD deliveries. Not surprisingly, this appears to have consequences relative to risk of childhood chronic diseases.CS has been associated with altered levels of immune cell populations, cytokines, and chemokines in neonates leading Cho and Norman  to suggeCS with the outcome of low bacterial diversity in the infant is reported to increase the risk of several immune-based diseases emerging in children including asthma , 113, atIn children who experience abuse, the developing immune system appears to become wired for dysfunctional responses. In the Nurses' Health Study II, Bertone-Johnson  found thWhile human immunological studies on diethylstilbestrol (DES) are limited compared with other health-related studies, there are reports suggesting that prenatally-exposed offspring are at a higher risk of immune-based disease. Overall DES daughters exposed in utero self-reported an increased risk of all immune-based diseases . Within specific categories, the women experienced more infectious diseases than non-DES exposed daughters . In a seThere are substantial data from animals suggesting that developmental exposure to alcohol produces DIT , 124 andP = 0.024) even after exclusion of effects of maternal smoking or atopic dermatitis [Remarkably, human studies are limited for low-level ethanol intake and DIT-related outcomes. Most studies following children exposed in utero to alcohol have focused on growth and behavioral outcomes , 129. Carmatitis .Two studies reported negative results for maternal alcohol intake and childhood asthma. Yuan et al.  examinedA case-controlled study in Ireland with infants born in 1994–2001 examined factors that are potentially involved with sudden infant death syndrome (SIDS) . McDonneA cadre of heavy metals has been examined for DIT and associated health risks in both children and adults. Among the most consistent observations with lead (Pb) are elevated risk of oxidative damage and a skewing toward Th2-driven responses with elevated levels of IgE. As an indicator of Pb's ability to produce misregulated inflammation, Pineda-Zavaleta et al.  found thThere are several suggested developmental risk factors for asthma. Among these, maternal smoking during pregnancy and exposure of the infant to environmental tobacco smoke (ETS) were identified by Selgrade et al.  as havinγ. As previously mentioned in the section on Pb, Lutz et al. [γ to IL-4 among children of smokers. While IL-4 levels were unchanged in the comparison of children with parental smokers versus non-smokers, reduced IFN-γ was associated with parental smoking and a dose response relationship appeared to exist. Therefore, the balance of IFN-γ to IL-4 was shifted toward the latter.Wilson et al.  reportedz et al.  reportedz et al.  for expoγ production), and altered innate immune (defective TLR-driven) responses upon vaccination with tetanus toxoid. These studies suggest that early-life exposure to smoking causes immune dysbiosis (targeted inappropriately exaggerated responses as well as suppression) that includes both an elevated risk of certain allergic diseases as well as potentially impaired responses to childhood vaccination. In keeping with many other DIT studies involving other risk factors, it also suggests that some human subpopulations are likely to have enhanced vulnerability for smoking-related DIT.Elevated risk of allergic diseases is not the only immune-based concern with early-life exposure to tobacco smoke. Kum-Nji et al.  reviewedDisrupted immune maturation is not the only pathway through which maternal smoking and ETS appear to affect later-life immune function. Wilhelm-Benartzi  found thPrenatal and early infant exposure to paracetamol (acetaminophen) has been associated with an increased risk of a variety of wheeze-associated disorders in the child including asthma. In the case of prenatal exposure, a study from Denmark examined 197,060 singletons born in northern Denmark in 1996–2008 . ParacetOne of the potential confounding factors is prevalence of infections and the use of antibiotics, which may coincide with administration of paracetamol . HeintzeFor potential infant use, McBride  argued tNAT2, Nrf2, and GSTP1). Shaheen et al. [Nrf2) and glutathione S-transferase (GST) polymorphisms within data from the Avon Longitudinal Study of Parents and Children. They found that maternal Nrf2 allelic differences had an effect on early gestation exposure to paracetamol and childhood asthma, while the presence of the GSTT1 allele was important in late gestational exposure to paracetamol [Evidence from several studies suggests that disruption of effective oxygen species regulation is a likely route to the elevated risk. Kang et al.  reportedn et al.  examinedacetamol . Taken tPesticides fall into several different chemical categories . However, humans are likely to be exposed to pesticide mixtures rather than to a single pesticide, and mixtures may result in unanticipated interactions among the pesticides at the molecular level . Human eThree epidemiological studies are significant in pointing to similar conclusions regarding prenatal pesticide exposure and later childhood neurodeficits. In the Columbia University study, Rauh et al.  found anAmong pesticides, the exposure risks not only involve childhood-onset conditions but also later-life-appearing diseases . Zhou et al.  found thThere is a suggestion that pesticide exposure may affect the risk of immune-driven NCDs. In the U.S. Agricultural Health Study, Hoppin et al.  found thCorsini et al.  recentlyPolychlorinated biphenyls (PCBs) are in a category of persistent organic pollutants (POPs) that can present human health challenges long after release into the environment. Stølevik et al.  examinedSignificantly for risk of NCDs, Grandjean et al.  found thThe impact of developmental exposure to perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) was examined in a prospective cohort birth study in the Faroe Islands . The resDIT and fetal programming are emerging as significant contributors not only to later-life immune dysfunction and misregulated inflammation but also to increased risk of NCDs and particularly chronic diseases. Given the present epidemic of NCDs, the interrelated comorbidities that exist among a myriad of chronic diseases, and the role of NCDs as the greatest cause of death worldwide, better preventative strategies are needed.Animal model research of DIT dates back several decades and helped to establish the fundamental characteristics surrounding early-life immune vulnerability for later-life disease. Recently, human DIT-related data have shown the relevance of the animal model information concerning dose sensitivity, subpopulation vulnerability, and health ramifications. While data gaps still exist for some categories of environmental risk factors , the way forward seems clear.Better identification of DIT risk and improved protection of age-, sex-, and genotype-based hypervulnerable subpopulations are needed. This may well require a different approach to safety testing. With the potential for epigenetic alterations to be produced in utero and inheritance of altered immune- and inflammation-related gene expression across generations, it is apparent that efforts to reduce the prevalence of NCDs need to focus on early life. Reducing the prevalence of DIT is an important first step in comprehensive efforts to reduce the prevalence and global impact of NCDs.


An important modern goal of plant science research is to develop tools for agriculturalists effective at curbing yield losses to insect herbivores, but resistance evolution continuously threatens the efficacy of pest management strategies. The high-dose/refuge strategy has been employed with some success to curb pest adaptation, and has been shown to be most effective when fitness costs (fitness trade-offs) of resistance are high. Here, I use eco-evolutionary reasoning to demonstrate the general importance of fitness trade-offs for pest control, showing that strong fitness trade-offs mitigate the threat of pest adaptation, even if adaptation were to occur. I argue that novel pest management strategies evoking strong fitness trade-offs are the most likely to persist in the face of unbridled pest adaptation, and offer the manipulation of crop colours as a worked example of one potentially effective strategy against insect herbivores. A primary concern in agriculture is crop yield loss due to insect herbivores. Many successful methods of pest management have been developed to reduce such losses, dominated in the past century by the use of chemical pesticides applied topically to crops (Ware and Whitacre Bacillus thuringiensis (Bt) bacteria . In this approach, farmers use pesticides (or grow Bt crops) on only a portion of their farmed land, allocating the remaining land to a refuge where herbivores are not exposed to pesticides, such that pests in the refuge do not experience natural selection for resistance. The crux of the strategy is that herbivores from refuge populations mate within populations exposed to pesticides and thus introduce susceptible alleles that prevent the otherwise inevitable rise in resistant pest genotypes. Furthermore, using high doses of pesticides makes resistance alleles functionally recessive, such that heterozygotes harbouring both resistant and susceptible alleles experience mortality as severely as susceptible homozygotes. Coupling refuges with high doses is in concept so promising that even governmental agencies have become involved by mandating that farmers create pesticide refuges to prevent regional pesticide resistance evolution that threatens crop yields  has been afforded by this strategy, summing across the entire agricultural landscape. The answer to this question largely depends on the topology of trade-offs associated with pest evolution.FigureAlternatively, consider the same agricultural scenario, but for which there is a strong trade-off between resistance and susceptibility Fig.B. Under An additional feature of pest management strategies eliciting strong fitness trade-offs is that it is unnecessary to ensure resistance is recessive. Without a fitness trade-off, it is very important that resistance is recessive, i.e., that heterozygotes not express resistance. Otherwise, novel resistance alleles could more easily become established in a population and spread to fixation. On the other hand, with strong fitness trade-offs, it makes little difference whether traits relevant to the pest management strategy are recessive, because the effects of pest phenotype on mortality are more symmetrical between the two crop regions. Neither of the genotypes can be considered the ‘resistant’ genotype, as both experience equal mortality.In summary, the specific topology of fitness trade-offs has a profound influence on the consequences of pest adaptation. As shown above, if fitness trade-offs are weak, then pest adaptation can substantially reduce mortality and render a pest management strategy ineffective. However, if fitness trade-offs are strong, overall pest mortality is largely independent of pest adaptation. Although the verbal model presented above should suffice under most circumstances to aid in the understanding and application of these ideas, I have included a formalized mathematical model in the online supplement that is presented in both discrete-trait and quantitative-trait forms to facilitate deeper exploration.E. coli, but novel mutations subsequently restore high fitness, breaking the fitness trade-off  that yield loss is related to tissue removal by herbivores only (i.e. not to diseases vectored by pests) and (ii) a linear relationship between pest mortality and yield loss. However, deviations from a linear relationship are known to exist on account of compensatory growth following herbivory for many crops  will likely not be as high as, say, mortality of susceptible pests on Bt crops, where mortality is often greater than 99%  as proof-of-concept research accumulates. However, a preliminary analysis of feasibility is useful here by evaluating current knowledge about how camouflage influences pests, how pests influence yield, and the costs and benefits of developing and using anthocyanin-producing crops.First, whether colour intercropping could actually reduce herbivory enough to substantially curb yield loss is an open and very important question. Depending on the crop, animal pests threaten to reduce crop yields between 9 and 36% Oerke , but 100A second line of analysis requires integrating the costs of developing novel crops and the physiological cost of anthocyanin production with the ecological benefit of anthocyanin presence (Kursar and Coley Taken together, these analyses suggest that colour intercropping might be highly effective at curbing yield losses, but caution that any single pest management method might not serve well as a long-term panacea. Hence, I propose that multiple pest management strategies, each evoking strong fitness trade-offs, could be employed simultaneously to increase overall efficacy. It is my hope that fitness trade-offs will inspire agricultural researchers to develop new pest control techniques focused on insect herbivores and other pests, including weeds and microbial diseases, both of which are known to exhibit rapid evolution to attempts at management (Thrall et al. mutual negative cross-resistance occurs when the evolution of resistance to either of two pesticides results in sensitivity to the other. The use of negative cross-resistance in the context of the refuge strategy has been discussed before due to its potential to curb resistance evolution (Pittendrigh et al. One such trade-off-based approach that could be employed alongside colour intercropping is to use pairs of pesticides that drive the evolution of mutual negative cross-resistance. Negative cross-resistance occurs when evolved resistance to one pesticide confers sensitivity to another (Peiris and Hemingway


Plants and microbes coexist or compete for survival and their cohesive interactions play a vital role in adapting to metalliferous environments, and can thus be explored to improve microbe-assisted phytoremediation. Plant root exudates are useful nutrient and energy sources for soil microorganisms, with whom they establish intricate communication systems. Some beneficial bacteria and fungi, acting as plant growth promoting microorganisms (PGPMs), may alleviate metal phytotoxicity and stimulate plant growth indirectly via the induction of defense mechanisms against phytopathogens, and/or directly through the solubilization of mineral nutrients , production of plant growth promoting substances , and secretion of specific enzymes . PGPM can also change metal bioavailability in soil through various mechanisms such as acidification, precipitation, chelation, complexation, and redox reactions. This review presents the recent advances and applications made hitherto in understanding the biochemical and molecular mechanisms of plant–microbe interactions and their role in the major processes involved in phytoremediation, such as heavy metal detoxification, mobilization, immobilization, transformation, transport, and distribution. Soil contaminated with heavy metals has become a serious worldwide problem due to geologic and anthropogenic activities. Heavy metals are non-degradable and thus persist indefinitely in the environment. As an alternative to physical and chemical methods, the use of hyperaccumulating plants and beneficial microbes has been a promising approach to clean up metal contaminated soils through extraction (phytoextraction), stabilization (phytostabilization), and/or transformation (phytovolatilization) process .+) release or by forming organic/amino acid-metal/mineral complexes; (ii) intracellular binding compounds ; (iii) electron transfer by enzymes in the rhizosphere ; and (iv) indirectly stimulating rhizosphere microbial activity , therefore enhancing phytoremediation efficiency .Root exudates and microorganisms are important components of rhizosphere ecology and play important roles in changing the bioavailability of metals and nutrients. Root exudates provide microbes an abundant source of energy and nutrients for microbes, and in return, microbes stimulate exudation from plant roots. In the co-evolutionary process, plants and their associated microbes coexist or compete for survival in the changing environment, and their relationships, either beneficial or detrimental are of significant importance for both partners. Root exudates are known to enhance mobility of metals and nutrients by (i) acidification due to proton . In metal rich natural (serpentine soil) and anthropogenic contaminated habitats , microbes are able to tolerate considerable high concentrations of metals, and to evolve resistance strategies . Microbes can enhance phytoremediation in different manners: expedite plant biomass, increase (phytoextraction) or decrease (phytostabilization) metal availability in soil, as well as facilitate metal translocation from soil to root (bioaccumulation) or from root to shoot tissues ; and (2) heavy metals versus plant–microbe interactions .The interactions between root exudates and microorganisms in the rhizosphere have been recognized as a critical component of plant growth in phytoremediation . HoweverTable 1. Root exudates play an important role in phytoremediation, as they induce the capability of host plants to actively adapt to and survive under metal stress conditions by either allelopathic functions (affecting the growth of rhizosphere microbes and other plants), or detoxification process . In addition, root exudates, particularly organic acids are able to bind metal ions, therefore influencing metal mobility, solubility and bioavailability in soil . Echinochloa crusgalli significantly enhanced both translocation and bioaccumulation of metals , suggesting that organic acids can be considered natural chelating agents to enhance phytoextraction. However, some components of root exudates do not influence metal availability or have a negative impact on metal mobilization . In the rhizosphere, plants can effectively communicate with their neighboring soil microorganisms by exuding chemicals or signals , while their associated microbes may establish an efficient associative symbiosis with plants by triggering host functional signals .In general, plants possess the ability to select their own root microflora from the surrounding soil and each particular plant species has a characteristic group of associated microbes . This prFigure 2).Extensive communication between plants and microbes via various signaling molecules plays a significant role in maintaining the growth of both partners. These include plant-released chemical signals that are recognized by the microbes and microbial signals and volatiles that trigger changes in plant physiology . Flavonoids play a significant role in AMF spore germination, hyphal growth, differentiation, and root colonization in AMF-plant interactions  genes involved in the synthesis of lipochitin–oligosaccharide signaling molecules, the Nod factors . Bacterial VOCs  can establish the communication with plants, trigger plant defense and growth promotion mechanisms by enabling host plants to colonize nutrient  poor soils . These functions can contribute to improve plant growth, which is fundamental for successful phytoremediation. Bacillus subtilis and B. amyloliquefaciens can activate ISR in Arabidopsis seedlings challenged with the soft-rot pathogen Erwinia carotovora subsp. carotovora. Moreover, some bioactive VOCs produced by PGPB such as ammonia, butyrolactones, hydrogen cyanide, phenazine-1-carboxylic acid, alcohols, etc. are able to affect mycelium growth and sporulation of different fungal species  were able to trigger germination and metabolism of Papaver somniferum  are able to alter the chemical composition of root exudates and thus plant physiology via releasing of various signaling molecules, such as volatile organic compounds (VOCs), Nod factors, Myc factors, microbe-associated molecular patterns (MAMPs) and exopolysaccharides . The QS induced processes such as sporulation, competence, antibiotic and biofilm production, have been widely documented in plant–microbe interactions (N-acyl-L-homoserine lactones (AHLs) are the essential components of this communication system. AHL quorum signals can enhance or inhibit diverse phenotypes depending on the bacteria being beneficial or pathogenic  and/or PGPB  released by rhizobia Sinorhizobium fredii, Rhizobium etli, and R. sullae were involved in interactions with their host legumes. Similarly, Arabidopsis thaliana roots with the bacterial QS molecule N-hexanoyl-homoserine lactone (C6-HSL) caused distinct transcriptional changes in legume tissues. Moreover, the AHL mimic compounds  secreted by higher plants  and other eukaryotic hosts can disrupt or manipulate QS-regulated behaviors among bacterial population (nod and rhizosphere-expressed (rhi) genes] in plant tissues  is controlled by diffusible signaling molecules produced by individual bacterial cells , which act as chemoattractant signals and/or sources of carbon and nitrogen for microbes . It has been established that plants can elicit crosstalk to microorganisms by secreting root exudates as signaling molecules, enabling colonization by some beneficial bacteria while inhibiting the other pathogenic bacteria . For microbial endophytes, one more step is required that is entry into plant tissue interior and formation of micro-colonies either inter- or intracellularly .Plant–microbial communication is a critical process to characterize the belowground rhizosphere zone, which can be beneficial to host plants and microbes. The metal resistant beneficial microbes (bacteria and AMF) are often used as bioinoculants to affect metabolic functions and membrane permeability of root cells and thus to enhance the establishment, growth and development of remediating plants in metal contaminated soils through: (1) facilitating mineral phytoavailability  by acting as biofertilizers; (2) modulating phytohormones balance by acting as phytostimulators; (3) reducing ethylene synthesis by acting as stress bioalleviators; (4) preventing deleterious effects of phytopathogens via production of antifungals and ISR, by acting as biopesticides; and (5) modifying root biomass and morphology by acting as biomodifiers . Examples are NFB  and nutrient-absorbing AMF, which can improve the fertility of polluted soils for plant growth by catalyzing the reduction of atmospheric N2 to biologically available ammonium  uptake by leguminous trees grown on Pb/Zn mine tailings. Further, P is one of the major macronutrients required for plant growth, however, most P compounds are not readily soluble in soil and are hence unavailable to plants  deficiency in soil. Recently, the role of siderophore producing microbes (SPMs) such as bacteria and fungi in Fe acquisition by different plant species as well as the mechanisms behind their promotion of Fe acquisition have been widely studied , Polaromonas sp. and Janthinobacterium sp. (Betaproteobacteria), were previously reported to accelerate the dissolution and mobilization of mineral nutrients  for soil fertility  in metal contaminated soils, therefore develop longer and prosperous root system and get better established during the early growth stage, which is highly desirable in heavy metal phytoremediation . The IAA synthesized by microbes, together with endogenous plant IAA, cannot only stimulate root exudation and proliferation of lateral and adventitious roots, but also induce the synthesis of ACC synthase  exert negative effects on bacterial IAA production. The IAA synthesized by RC6b induced significantly greater root growth of Sedum plumbizincicola than that of non-inoculated control plants. Furthermore, AMF colonization also has positive effects on plant cell growth and division as a result of fungal hormones production. Glomus intraradices and Gigaspora margarita onto seedlings of Litchi chinensis significantly increased the IAA and isopentenyl adenosines concentrations in shoots and roots. The changes in endogenous phytohormones level were responsible for morphological alteration induced by AMF inoculation.Plant associated microbes can also produce phytohormones such as IAA, cytokinins, gibberellic acid, abscisic acid and others, even under stress conditions, thereby modulating the hormonal balance in plants and their response to stress . One of the key traits related with plant growth promotion is the production of ACC deaminase by PGPB that hydrolyses the plant ethylene precursor ACC into ammonia and α-ketobutyrate  and roots (from 35 to 123%), compared to the non-inoculated control (Brevibacillus laterosporus produced two extracellular chitinase (89.6-kDa four domain and 69.4-kDa two domain) that contribute to its antifungal and insecticidal activities. In addition, some exopolysaccharides-producing plant growth promoting rhizobacteria (PGPR) such as Proteus penneri, P. aeruginosa, and Alcaligenes faecalis were proved to alleviate water stress and improve plant biomass under drought stress  and rhizobitoxine; butyrate . The ino control . ACC dea control . Rhizobi control . Prasannt stress .Achromobacter, Azospirillum, Burkholderia, Methylobacterium, and Psychrobacter can increase some root morphological traits .The discovery of plant-microbe-metal interactions sustains the importance of plant–microbe interactions in the biogeochemical cycling of metals and in their application in phytoremediation. It is essential to consider the appropriate combination of plants and microbes involved in applied processes for enhanced phytoremediation efficiency, in order to obtain the best performance from the existing microbe-based technologies. The plant-microbe-metal interactions spanning from both macropartner (higher plants) and micropartner (microorganism) to heavy metals is nevertheless a crucial step in understanding plant metal uptake during geo-bio-interactions . Among the tolerance mechanisms employed by metal-accumulating plants, the exudation of various compounds, especially LMWOAs is able to stimulate microbial growth, solubilize insoluble or sparingly soluble mineral nutrients , and detoxify some metals  and metallothioneins (MTs)], as well as sequestration of metal-siderophore complexes in root apoplasm or soils  cell surface biosorption/precipitation of metals; (2) active eﬄux pumping of metals out of the cell via transporter system; (3) sequestration of metals in intracellular compartments (mainly cell vacuole); (4) exclusion of metal chelates into the extracellular space; and (5) enzymatic redox reaction through conversion of metal ion into a non-toxic or less toxic state.Moreover, plant associated AMF and bacteria are capable of inducing metal translocation and distribution, thus metal allocation to the inner root parenchyma cells . The mecFigure 3). Bioaccumulation is a process of intracellular accumulation of metals. It comprises two stages: metabolism-independent passive biosorption , and metabolism-dependent active bioaccumulation  and bacterial inoculation ameliorated metal toxicity through biosorption, thus exhibiting a protective effect on host plant growth. Dissimilarly, Aspergillus and Rhizopus and found no direct relationship between metal tolerance and biosorption properties of these fungi. The bioaccumulation process is more complex than biosorption and it requires metabolic activity of living cells involving intracellular sequestration (MTs and PCs binding), extracellular precipitation, metal accumulation and complex formation  contributes to entrap metal ions either in living or dead cells, whereas bioaccumulation through helper proteins is involved in the incorporation of essential nutrients  or living (bioaccumulation) cells ] and iron-oxidizing bacteria  and fungi . Acidithiobacillus thiooxidans created acidic conditions favorable for bioleaching/removal of metals . The capability of bioleaching depends on bacterial species. In general, acidophilic bacteria are more capable for metal bioleaching than neutrophilic bacteria . Non-essential metals such as Cd and As, generally enter the cell through either non-ATPase or ATPase-linked nutrient transport systems that are highly specific for the imported cation or anion (2+) from bacterial cells can be achieved through an ATPase eﬄux mechanism .It is well-known that strong binding of metals to soil particles or precipitation accounts for the insolubilization of a significant fraction of metals in soil and consequently contributes to their unavailability for plant uptake. The mobility and solubilization of metals in soil have been recognized as critical factors in affecting the practical efficiency of phytoextraction , 2013. ISphingomonas SaMR12 regulated quantity of root exudates (organic acids) from S. alfredii, thus substantially improving Cd bioavailability and plant absorption facility. A recent study reported that P. myrsinacearum RC6b significantly increased metal uptake by S. plumbizincicola. This was attributed to its ability to produce organic acid and solubilize insoluble tricalcium phosphate . In order to understand, predict and optimize such processes, there have been extensive attempts to model the interactions between protons, metal ions and bacterial surfaces, as well as to characterize them using spectroscopy. 2+ with surface protonable groups of Rhodobacter sphaeroides by using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The results revealed that carboxylate moieties on the bacterial surface play a significant role in extracellular biosorption of Ni2+, establishing relatively weak bonds with metal ion.Soil microbes can also acidify their environment by exporting protons to replace heavy metal cations at sorption sites . In contrast to PCs, MTs, small cysteine-rich and metal-binding proteins, play essential roles in activities of various organisms  such as metal detoxification and homeostasis through scavenging reactive oxygen species (Tuber melanosporum polypeptides such as MTs (TmelMT) and PC synthase (TmelPCS) were capable of conferring an enhanced tolerance to essential (Cu and Zn) and non-essential  thiophilic metal ions in yeast. Nevertheless, it is known that MTs also occur in AMF and that genes encoding several enzymes for PCs synthesis can be activated in mycorrhizal roots, therefore enhancing photosynthetic activity in mycorrhizal plants exposed to metal stress. However, these metal chelation mechanisms cannot make a major contribution to metal tolerance strategies operating in AM symbiosis  can eliminate the phytotoxic effect of free metal ions, allowing for metal uptake, sequestration, compartmentation, xylem loading, and transport within the plant . Phytoch species . Bolchi ymbiosis .3+-PS]; and Strategy III . Many studies have shown that PSs are able to solubilize and transport metals by chelation, and thus being secreted into the rhizosphere through a potassium-mugenic acid symporter . Recently, Rahnella sp. JN27 promoted Cd solubilization in metal-amended soils through the release of siderophores, therefore facilitating Cd uptake by Cd-hyperaccumulators Amaranthus hypochondriacus and A. mangostanus.Fe is an important micronutrient and its concentration in soil is often below the level necessary to support robust plant and microbial life due to its low solubility, especially under metal stress. Therefore, plants surmount challenges to acquire sufficient Fe through three mechanisms, namely, Strategy I ; Strategy II [secretion of phytosiderophores (PSs) and uptake Feymporter . It has Figure 3). Organic acids excreted by plant roots, such as malate, citrate, acetate, and oxalate, are widely recognized to be responsible for dissolving the solid phase metals in soil through complexation reaction and thus making them available for plant uptake . Juncus maritimus acted as complexing agents of trace metals, and were responsible for the enhanced metal mobility and availability in soil. However, the excretion of organic acids by microorganisms has a somehow more profound effect on the stimulation of metal release than the direct effect of root secretions  and metal availability by accelerating host-mediated LMWOAs secretion, thereby significantly enhancing shoot biomass production of Solanum nigrum and accumulation of metals  in aerial plant parts. Nevertheless, AMF have not been shown to produce organic acids. However, the specific protein glomalin produced by AMF seems to be efficient in sequestering heavy metals outside the mycelium . The structures of BSs are usually composed of one or more compounds, such as mycolic acid, glycolipids, polysaccharide-lipid complex, lipoprotein or lipopeptide, phospholipid, or the microbial cell surface itself  are amphiphilic compounds either produced on microbial cell surfaces or excreted extracellularly, and contain hydrophobic and hydrophilic moieties that reduce surface and interfacial tensions . Azotobacter vinelandii bound metal cations and oxoanions in its extracellular medium, therefore increasing the metal (Fe and Mo) bioavailability to bacterial cells, which can subsequently recruit the complexes. The efficient quantification of those metal complexes is crucial for essential cofactors of nitrogenase  homeostasis, N2 fixation dynamics and N2 cycle. This strategy allows microbes to control metal speciation in their environment, thereby increasing heavy metal bioavailability to the N2 fixers.Metallophores are low molecular weight organic ligands released from microorganisms, which can regulate the intracellular metal concentrations to avoid toxicity or maintain appropriate concentrations for their growth .Some microbes can also reduce plant metal uptake or translocation to aerial plant parts by decreasing metal bioavailability in soil via precipitation, alkalinization, and complexation processes  and toxic metals  by microbial reduction processes, which show considerable promise for phytoremediation of metal contaminated soils . Oves etc action , or indic action . The micc action . Similar-, can result in an active nitrate uptake by microbes and a reduction in metal phytoavailability in the rhizosphere by secreting glomalin  exhibit the ability to absorb metals through substratum alkalinization activity, therefore affecting the metal stability in soil . The alkglomalin . AMF canglomalin . Hu et aFigure 3). The mechanisms involved in metal biosorption onto to EPS include metal ion exchange, complexation with negatively charged functional groups, adsorption and precipitation . In general, metals such as Cu and Hg are more soluble in their lower oxidation state, whereas the oxidation states of metals such as Cr, As, and Se are more soluble and toxic . Due to the high cation exchange capability of the xylem cell, the metal movement is severely retarded when the metals are not chelated by ligands. Organic acids are involved in the Cd translocation in Brassica juncea  in soil and significantly increased metal accumulations (Cd and Zn) in shoots of S. plumbizincicola. Besides, a few studies found that the growth and metal accumulation in the above ground part of plants have been improved by using combinations of plant associated microorganisms. Salix viminalis x caprea with ectomycorrhiza associated bacteria Micrococcus luteus and Sphingomonas sp. in combination with the fungus Hebeloma crustuliniforme increased the total Cd and Zn accumulation in shoot up to 53 and 62%, respectively, and consequently led to an increase in phytoextraction of Cd and Zn in these fungal-bacterial inoculant combinations. Similarly, Streptomyces sp. and Agromyces sp. plus the fungus Cadophora finlandica onto Salix caprea, shoot concentrations of Cd and Zn were mostly increased, indicating higher translocation of metals from roots to shoots.The translocation of metals from plant roots to the above-ground parts varies considerably depending on plant species and metals. Different metals are differently mobile within a plant, e.g., Cd and Zn are more mobile than Cu and Pb. During the transportation through the plant, metals are bound largely on the cell walls of roots, leading to high metal concentration in plant roots. The metal chelation with ligands  facilitates the metal movement from roots to shoots  providing deep insights on biochemical and molecular mechanisms of plant–microbe interaction, which could contribute to evolution dynamics of microbial consortia; (2) demonstrating the effectiveness of microbes devoted to hold potential stress-reducing properties and conferring their host plants metal stress resistance by acting as bioprotectants; (3) improving knowledge of how beneficial plant–microbe association may contribute to develop microbial inoculants and to promote plant yield respecting ecosystem biodiversity and safety by acting as biofertilizer; and (4) elucidating the mechanisms of cooperative plant–microbe interactions during metal detoxification, mobilization, immobilization, accumulation, translocation and transformation, which could contribute to the implementation of phytoremediation technologies.The future research should be focused on: (1) the mechanism of plant-microbe-metal interactions under stressful environmental conditions; (2) the effectiveness of co-inoculation of PGPB and AMF response to multiple biotic and/or abiotic stress; (3) the identification of functional genes of beneficial microbes for growth enhancement and metal metabolism; (4) the optimization of techniques for application in large scale polluted fields; and (5) the exploration of commercial production of bioinoculants for use in metal decontamination.YM developed the ideas and wrote the manuscript; RO and CZ revised the manuscript; HF was the project sponsor.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gaucher disease is an autosomal recessive systemic condition, and the most common of the lysosomal storage disorders. It is characterized by lipid accumulation in certain cells and organs, particularly macrophages, which appear on light microscopy as ’Gaucher cells’ or vacuolated lipid-laden reticuloendothelial cells. Long bone involvement is common in Gaucher disease, whereas craniofacial bone involvement is extremely rare. Reports confirming the diagnoses of Gaucher disease involving craniofacial bones by histopathologic evidence are even rarer.A 46-year-old Caucasian Ashkenazi Jewish woman with Gaucher disease presented with jawbone pain and lytic radiographic lesions of her mandible. Surgical biopsy of a mandibular lesion revealed Gaucher cells infiltrating the mandible, which correlated with radiographic and clinical findings, supporting a diagnosis of Gaucher disease with jawbone involvement.Lysosomal storage diseases can have head and neck manifestations, and bone involvement in Gaucher disease is common. Therefore, careful consideration of signs and symptoms and medical history, with a thorough review of systems, is important when evaluating patients with lysosomal storage disorders to rule out head and neck involvement of disease. Biopsy may be warranted in some cases for more definitive diagnosis of painful jawbone lesions and to rule out other odontogenic and non-odontogenic conditions in the differential diagnosis. Gaucher disease is an autosomal recessive systemic lysosomal storage disorder that is panethnic but with higher prevalence in the Ashkenazi Jewish population. The disease is caused by a defect in the housekeeping gene lysosomal glucocerebrosidase on the first chromosome 1q22) [q22 [1]. Infiltration of Gaucher cells in tissues can be associated with systemic pathology such as hepatosplenomegaly, pancytopenia, skin pigmentation, neurologic symptoms, osteoporosis and severe bone pain . Oral paGiven the potential association with teeth, the clinical nonspecific presentation of pain, and the aforementioned radiographic features which could represent several odontogenic or non-odontogenic conditions, a diagnosis of Gaucher disease involving the jawbone can be diagnostically challenging and may warrant biopsy for definitive diagnosis. Only a few reports in the literature provide definite diagnosis of jawbone involvement by Gaucher disease as confirmed by biopsy rather than clinical and radiographic findings alone ,8-14. WeA 46-year-old Caucasian woman of Ashkenazi Jewish descent presented with the chief complaint of severe and constant throbbing pain in her left posterior mandible. She reported that her pain was aggravated by chewing, and started months before the initial consultation visit. Visual analog scale of pain was reported 7/10 at the day of the visit. Past medical history was remarkable for Gaucher disease, left hip replacement, gall bladder removal, herniorrhaphy, spleen abscess drainage, hepatitis C infection, and asthma. Social history was significant for 20 pack-years of cigarette smoking. Her family history was unremarkable for any hereditary or systemic diseases. Her medications included the oral antiresorptive drug alendronate for early osteoporosis associated with Gaucher disease (70mg weekly for 2 years), calcium supplements, loratadine, hydromorphone, esomeprazole, promethazine, levalbuterol, carisoprodol, fluticasone and ibuprofen. She reported that she had been on enzyme replacement therapy (ERT) intermittently for several years to treat her Gaucher disease which was diagnosed 20 years prior. She received intravenous injections twice a month for ERT and recounted fatigue after each infusion. She also reported receiving antibiotic and analgesic medications for her pain months ago but they were not helpful.Head and neck examination, cranial nerve examination and vital signs were within normal limits. Intraoral examination was completed using percussion testing, vitality testing and probing for evaluation of her mandibular posterior teeth in the region of the chief complaint. Her left mandibular first molar was endodontically treated 3 years prior and tested non-vital, while her remaining mandibular teeth were normal on vitality testing with evaluation of several opposing non-restored and non-carious teeth as internal controls. Gingival tissues in both jaws were non-inflamed and there was no gross evidence of plaque or calculus. Periodontal pockets in her mandible ranged from 4 to 6mm with no significant bony defects. Full mouth dental X-rays and a panoramic radiograph were taken and showed relatively well-defined radiolucent lesions in multiple regions of the mandible bilaterally, with a pseudocystic (multilocular) appearance; cone-beam computed tomography confirmed these findings and revealed a large lytic lesion of her left posterior mandible in the region of her chief complaint (Figure Patients with Gaucher disease often complain of severe pain in various parts of their skeletal system, but rarely in the craniofacial or jawbones. Bender and Bender reported two cases of Gaucher disease with 13- and 60-years follow-up; in the first case mandibular lesions were present in the premolar-molar region and the involved teeth were vital . These fet al. [In the current case, radiographs of the head and neck showed lytic radiolucencies bilaterally in her mandible. Although the maxilla or sinuses can be involved in Gaucher disease, we could not appreciate such involvement in this case. A comprehensive review of the many radiographic jaw features in Gaucher disease was recently reported by Zeevi et al. . Some ofThe key to diagnosis of Gaucher lesions in the jawbones is thorough medical history and clinical examination, vitality and percussion testing of associated teeth when applicable to rule out odontogenic infections or lesions, radiographic evaluation, and in some cases histopathologic examination for definitive diagnosis.Lysosomal storage diseases can have head and neck involvement, and bone involvement in Gaucher disease is common. However, craniofacial bone involvement such as the jawbones is rarely reported. When involving the jawbones, Gaucher disease can mimic other odontogenic and non-odontogenic diseases and can be a diagnostic challenge. Therefore, familiarity with Gaucher disease and radiographic features in the jaws can facilitate accurate clinical diagnosis and management. Careful consideration of signs and symptoms and medical history, with a thorough review of systems, is important when evaluating patients with lysosomal storage disorders to rule out head and neck involvement of disease. Biopsy may be warranted in some cases for more definitive diagnosis of painful jawbone lesions since pain is a rare feature of Gaucher disease affecting the jawbones, and to rule out other odontogenic and non-odontogenic conditions in the differential diagnosis.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.All authors evaluated the patient, collected data, wrote and approved the final manuscript.


Anemia is a multifactorial condition whose prevalence increases in both sexes after the fifth decade of life. It is a highly represented phenomenon in older adults and in one-third of cases is “unexplained.” Ageing process is also characterized by a “multiple hormonal dysregulation” with disruption in gonadal, adrenal, and somatotropic axes. Experimental studies suggest that anabolic hormones such as testosterone, IGF-1, and thyroid hormones are able to increase erythroid mass, erythropoietin synthesis, and iron bioavailability, underlining a potential role of multiple hormonal changes in the anemia of aging. Epidemiological data more consistently support an association between lower testosterone and anemia in adult-older individuals. Low IGF-1 has been especially associated with anemia in the pediatric population and in a wide range of disorders. There is also evidence of an association between thyroid hormones and abnormalities in hematological parameters under overt thyroid and euthyroid conditions, with limited data on subclinical statuses. Although RCTs have shown beneficial effects, stronger for testosterone and the GH-IGF-1 axis and less evident for thyroid hormones, in improving different hematological parameters, there is no clear evidence for the usefulness of hormonal treatment in improving anemia in older subjects. Thus, more clinical and research efforts are needed to investigate the hormonal contribution to anemia in the older individuals. Anemia is a multifactorial condition whose prevalence increases in both sexes after the fifth decade of life, becoming a highly represented phenomenon in older adults . The estIn the elderly, anemia is caused by different factors including nutrient deficiencies , occult hemorrhage, renal dysfunction, and chronic inflammation/diseases . HoweverAlthough mild anemia can be well tolerated in young and adult individuals, this condition in the elderly is a predictor of a progressive decrease in physical performance and muscle strength , 11, andMoreover, large population-based prospective studies performed in community-dwelling older individuals identified anemia as risk factor for death, independently of multimorbidity , 18. An Efforts to understand the pathophysiology of the so-called anemia of aging have specifically targeted different physiological age-related changes including erythropoietin (EPO) resistance and reduced proliferative capacity of bone marrow stem cells, stem cell aging, impaired renal function, myelodysplasia (MDS), and chronic inflammation with higher circulating levels of proinflammatory cytokines , 23, 24.Among the predisposing factors of anemia recent studies underline the role of hepcidin, an iron-regulating hormone, which at higher levels seems to favour both iron sequestration in reticuloendothelial cells and the alteration of gut ferroportin-mediated iron absorption . HoweverInterestingly, the age-related decline in androgen levels has been recently numbered among the factors involved in the pathogenesis of the so-called UA of the elderly , 28. EpiTherefore, anabolic hormones, whose levels undergo a progressive decline during ageing, might represent a plausible cause of anemia when other causal pathways have been excluded. Interestingly, the ageing model is frequently characterized by a simultaneous deficiency in anabolic hormones. Multiple hormonal “dysregulation” is a more frequent phenomenon than a single hormonal derangement in older persons . This phA multiple hormonal derangement, rather than a single anabolic hormone deficiency, has been identified as a robust biomarker of health status in older persons . Data frAccording to this concept a partial failure of a single hormone system might be compensated by one or more parallel systems without producing significant detrimental clinical effects, suggesting that the single hormonal derangement is a not specific predictive parameter in older individuals.Data were especially focused on the erythropoietic properties of a single anabolic agent , with limited evidence existing on the contribution of multiple anabolic pathways in the pathogenesis of anemia.Thus, we performed an accurate review of the literature using PubMed until March 2015, by selecting the observational human studies investigating the hormonal contribution  to anemia older subjects of both sexes. In this review, studies are presented according to the cross-sectional and longitudinal design and to the positive or neutral effects on different hematological parameters. We also analyzed the clinical trials having hematological indexes as the main or secondary outcome.The role of androgens on erythropoiesis is known since 1941  when, beIn vitro and animal studies have suggested a role for androgens in increasing erythroid mass, EPO synthesis, and iron bioavailability. In bone marrow cultures, androgens stimulate hematopoiesis . Th. Th88]. er week) .By contrast, a double-blind, placebo-controlled study of GH treatment in elderly patients on chronic hemodialysis did not show any effect of GH on Hb levels . HoweverFinally, several trials were designed in order to address the effect of EPO replacement therapy on anemia and IGF-1. However, the effect of EPO therapy on IGF-1 serum level is still debated. In uraemic patients, EPO administration led to an enhancement of the GH response to the growth hormone releasing hormone (GHRH) . In hemoThe relationship between thyroid diseases and erythropoietic dysfunctions has been documented in experimental and human studies . In bothTSH could affect hematopoiesis by binding to a functional thyrotropin receptor (TSHR), which is found in both erythrocytes and some extrathyroidal tissues . Interes in vitro evidence that both T3 and T4 are implicated in the regulation of hematopoiesis by influencing the erythroid precursor proliferative capacity  [Similarly, Cinemre at al. showed that the T4 coadministration with iron  levels, and GH/IGF-1 axis) and erythropoiesis.Anabolic hormonal pathways could be linked to each other to some extent in influencing different steps of the erythropoietic process. However, very few studies have investigated the potential interaction between anabolic hormones  [2 levels were directly associated with hematocrit [The interrelationship between serum T, SHBG, and E88–1991) . In thismatocrit . Neverthmatocrit .α) synthesis [α [Some of T activities on erythropoiesis could also be linked to the GH/IGF-1 anabolic pathway . There iynthesis –243. Acchesis [α , suggestIn the last five decades, the erythropoietic role of anabolic hormones on anemia has been particularly investigated in a large number of studies. Different molecular mechanisms by which androgens, IGF-1, and thyroid hormones can affect the hematopoietic process have been identified. However, the interaction between different anabolic hormonal pathways and the contribution of multiple hormonal dysregulation to the pathogenesis of the mild anemia of the elderly has been poorly addressed.A very limited number of epidemiological studies have tested the role of the age-related decline in anabolic hormones as contributing factor of anemia, especially in older individuals. Notwithstanding the fact that RCTs testing the effects of a single hormonal replacement therapy on different hematological parameters yielded more convincing results, these studies have been focused on cohorts with a wide range of age and multimorbidity distribution. Moreover, hematological indexes were not the primary outcomes in all cases.In particular, Hb levels have been significantly correlated with the severity of androgen deficiency and T status. The RCTs and registries testing the effects of T therapy on hematological parameters have shown that erythrocytosis is the most common adverse event of testosterone replacement therapy. However, the usefulness of T therapy in anemic men exhibiting an age-related hypogonadism remains to date a controversial issue. The balance between long-term clinical benefits and risks of T replacement therapy, especially at prostatic and cardiovascular level, has not been adequately assessed in large, randomized clinical trials involving older men. IGF-1 has been hypothesized as cause of anemia under pathological conditions including a wide range of anemic disorders in observational studies. IGF-1 is a regulator of erythropoiesis in children with short stature, adults with GH deficiency, and those affected by chronic kidney diseases. A positive and significant correlation between Hb concentration and both serum IGF-1 and IGFBP-3 levels has been also observed in few studies conducted on healthy prepubertal and early pubertal subjects, nondiabetic adult subjects, and older individuals. Although intervention studies testing the effect of GH therapy on indexes of erythropoiesis in several diseases seem to support the beneficial effect of GH administration on anemia, there is limited evidence in healthy individuals.Finally, a large body of observational evidence suggests an association between overt thyroid hormones anemia and erythrocyte abnormalities. However, there is limited evidence targeting subclinical thyroid conditions, which are more frequently observed in the older population.Very few intervention studies have tested the effects of thyroid hormones replacement therapy on Hb concentrations and erythrocyte parameters, with most of them showing no beneficial effect of levothyroxine treatment.In conclusion, despite the biological rationale for considering the decline in anabolic hormones and alteration in thyroid hormones as determinants of mild anemia of aging, the usefulness of hormonal replacement therapy for treating anemia remains to date a controversial issue.