Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders

Pascal Andujar
Angélique Simon-Deckers
Françoise Galateau-Sallé
Barbara Fayard
Gregory Beaune
Bénédicte Clin
Marie-Annick Billon-Galland
Olivier Durupthy
Jean-Claude Pairon
Jean Doucet
Jorge Boczkowski
Sophie Lanone
Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders
R E S E A R C H Open Access
Background: Although major concerns exist regarding the potential consequences of human exposure to nanoparticles (NP), no human toxicological data is currently available. To address this issue, we took welders, who present various adverse respiratory outcomes, as a model population of occupational exposure to NP. The aim of this study was to evaluate if welding fume-issued NP could be responsible, at least partially, in the lung alterations observed in welders. Methods: A combination of imaging and material science techniques including ((scanning) transmission electron microscopy ((S)TEM), energy dispersive X-ray (EDX), and X-ray microfluorescence (μXRF)), was used to characterize NP content in lung tissue from 21 welders and 21 matched control patients. Representative NP were synthesized, and their effects on macrophage inflammatory secretome and migration were evaluated, together with the effect of this macrophage inflammatory secretome on human lung primary fibroblasts differentiation.Results: Welding-related NP (Fe, Mn, Cr oxides essentially) were identified in lung tissue sections from welders, in macrophages present in the alveolar lumen and in fibrous regions. In vitro macrophage exposure to representative NP (Fe 2 O 3 , Fe 3 O 4 , MnFe 2 O 4 and CrOOH) induced the production of a pro-inflammatory secretome (increased production of CXCL-8, IL-1ß, TNF-α, CCL-2, −3, −4, and to a lesser extent IL-6, CCL-7 and −22), and all but Fe 3 O 4 NP induce an increased migration of macrophages (Boyden chamber). There was no effect of NP-exposed macrophage secretome on human primary lung fibroblasts differentiation.Conclusions: Altogether, the data reported here strongly suggest that welding-related NP could be responsible, at least in part, for the pulmonary inflammation observed in welders. These results provide therefore the first evidence of a link between human exposure to NP and long-term pulmonary effects.

Background

Nanotechnologies represent a major stake of the 21 rst century. They are aimed at conceiving, characterizing and producing materials at the scale of the billionth meter; the nanometer. These nanomaterials (among which are particles with a diameter lesser than 100 nm -nanoparticles, NP) present new properties that make them particularly interesting to industrialists. The list of actual uses and applications for nanomaterials is already substantial, and will certainly become exponential in the next future. Such developments, in spite of potential benefits in numerous domains, i.e. in the field of nanomedicine, are accompanied by concerns regarding the potential consequences of human exposure (in the general population or in an occupational context), with particular threat at the respiratory level [1][2][3]. The need for toxicity evaluation is motivated by numerous studies already available in the literature demonstrating that animal exposure to engineered NP is followed, among other pathological manifestations, by the induction of a pulmonary inflammation, associated with lung remodelling [4][5][6][7][8]. However, so far, there is almost no study assessing the consequences of human exposure to manufactured NP. This point is critical since human exposure studies constitute the cornerstone of risk evaluation for health after (chronic) exposure to environmental agents, especially in the occupational context.

During their everyday occupation, welders are exposed to a complex aerosol of gases (e.g. carbon monoxide, ozone), and hazardous metal fumes composed of chain-like agglomerates of particles, with a primary size in the nanometer size range [9]. Indeed, up to 11% of the total mass, and 80% of the total number of particles emitted in welding fumes are NP [10,11]. Various adverse respiratory outcomes have been described in welders, among which inflammation and lung remodeling are largely described [12][13][14][15][16]. However, the underlying responsible mechanisms remain to be elucidated.

We therefore made the hypothesis that these abnormalities could result, at least partly, from an inflammatory effect directly induced by welding fume-issued NP present in the pulmonary tissue. Such hypothesis has never been studied in the literature so far, probably due to the technical challenges related to the analysis and characterization of NP tissular content in biological samples in situ. We therefore setup an original interdisciplinary study, combining the expertise of material science physicists, physico-chemists, biologists and medical doctors. A broad range of imaging and material science techniques (scanning/transmission electron microscopy (S/TEM), energy dispersive X-ray (EDX), and X-ray microfluorescence (μXRF)), was used to characterize the association between lung histological modifications and the presence of NP in 21 well-documented arc welders and matched unexposed individuals. Since this analysis revealed the presence of NP in macrophages in the alveolar lumen and in areas of fibrous tissue in welders, our physico-chemist partner chemically synthesized NP representative of those identified in pulmonary tissue (Fe 2 O 3 , Fe 3 O 4 , MnFe 2 O 4 and CrOOH), to investigate their effects (in combination or not with exposure to cigarette smoke, as the majority of patients were smokers) on human macrophages. These exposures were performed in combination or not with exposure to cigarette smoke, as the majority of patients were smokers. Our results show that exposure to welding-related NP induce the production of a pro-inflammatory secretrome (increased production of CXCL-8, IL-1ß, TNF-α, CCL-2, −3, −4, and to a lesser extent IL-6, CCL-7 and −22). Moreover, all but Fe 3 O 4 NP induce an increased migration of macrophages.Finally, there was no effect of macrophage pro-inflammatory secretome on human primary lung fibroblasts differentiation. These results provide therefore the first evidence of a link between human exposure to NP and long-term pulmonary effects.

Results

Clinical and histological characterization of patients

The clinical characteristics of the 42 patients included in our study are given in Table 1. Consistent with the population studied, female were significantly less represented in the welder group as compared to controls (p = 0.038). Otherwise, consistent with the selective matching of controls with welders, the two populations were similar for age and tobacco history.

CD68 staining revealed the presence of a higher number of macrophages in lung tissue sections from welders as compared to control patients, localized in the alveolar lumen as well as in the fibrous tissue ( Figure 1). Importantly, black spots corresponding to aggregates of particles were essentially colocalized with CD68 staining suggesting a macrophagic internalization of these aggregates. The iron load, as revealed by Perls staining (Figure 1) and the quantification of siderophages (iron-laden macrophages) and ferruginous bodies (formed by macrophages) ( Table 2), were significantly higher in welders as compared to control patients. Finally, lung fibrotic lesions were identified in both control and welders, but were significantly more severe in welders as compared to control patients irrespective of the histological localization ( Figure 1 and Table 2). There was no difference for other respiratory lesions such as respiratory bronchiolectasis, bronchiolar dysplasia or metaplasia, or bronchiolitis between the two groups ( Table 2).

Patients' particle burden characterization Elemental mapping in lung tissue sections

We first wanted to get a general overview of the elemental mapping at the global level of the tissue. To that matter, we used synchrotron-based X-ray microfluorescence (μXRF) and determined the spatial distribution of elements performing 100 × 100 μm maps. Figure 2A shows typical elemental μXRF maps obtained for sulfur (S), iron (Fe), and manganese (Mn) in control patients and welders. Since S represents a characteristic element of biological materials, the shape of the tissue can be drawn by the S map [17]. Using this approach, we observed a significant overload of Fe, Mn, chromium (Cr) essentially and to a lesser extent for titanium (Ti) in welders as compared to control patients ( Figure 2B). This signal was essentially localized in the fibrous tissue ( Figure 2), and to a lesser extent in the alveolar lumen (data not shown). Remarkably, we could observe that iron-rich zones were often co-localized with other element-rich zones, particularly Mn and Cr ( Figure 2 and data not shown).

NP identification in lung tissue homogenates

To determine individual exposure to asbestos bodies and non fibrous mineral particles, a mineralogical analysis of lung tissue homogenates was performed. These experiments confirmed the metallic overload in welder's lung and identified a significantly higher mass concentration of total mineral and especially metallic particles in welders as compared to control patients (Table 3). Consistent with the initial selective matching of controls with welders, the two populations were similar for asbestos exposure characteristics. Finally, in order to characterize the size and chemical nature of the particles present in the pulmonary samples, we used a combined STEM-EDX (Scanning Transmission Electron Microscopy (STEM)-Energy Dispersive X-Ray (EDX)) analysis of lung tissue homogenates, and demonstrated that the particles are of nanometric size (20-25 nm individual diameter -STEM analysis Figure 3A), and confirmed the chemical nature (Fe, Mn, Cr essentially) and the co-localization of the chemical signals (EDX analysis, Figure 3B).

NP identification in lung tissue sections

Following the detection of NP in the lung tissue homogenates, we questioned the identification of these NP in situ at the nanometer scale and characterized their localization in lung tissue sections from patients, especially in areas of lung remodeling. We therefore proceeded to a combined TEM-EDX analysis of lung tissue sections and observed that, in comparison with control patients, large quantities of NP were localized in macrophages residues, and was present in the fibrous tissue of welders ( Figure 4). In accordance with the results obtained in lung tissue homogenates, these NP presented a spherical shape, and a diameter centered around 20-25 nm. The chemical nature of the complex metal oxide NP in the welders lungs were mainly Fe, Cr and/or Mn (and aluminium (Al), silicium (Si), and nickel (Ni) to a lesser extent), whereas it was essentially Al and Si NP in the control patients (data not shown).

Overall, these different material science techniques allow us to demonstrate the presence of Fe, Cr and/or Mn NP in situ in lung tissue from welders, especially in macrophages.

Synthesis of representative NP

In order to answer the question whether the metal NP identified above have a causal role in the development of pulmonary alterations observed in welders, we next chemically synthesized 4 NP representative of those found in lung tissue samples; Fe 2 O 3 , Fe 3 O 4 , MnFe 2 O 4 and CrOOH. As shown in Figure 5, all NP were spherical and presented an average diameter of 25 nm (Fe 2 O 3 , Fe 3 O 4 , MnFe 2 O 4 ) or 15 nm (CrOOH) ( Figure 5A-D). The NP nature was verified by X-ray diffraction spectra ( Figure 5E-H).

The in vitro effect of the synthesized NP on the macrophage secretome

Because NP identified in lungs from welders were in macrophages, we first evaluated the effects of human macrophage exposure to the chemically synthesized representative NP in terms of inflammatory secretome. These experiments revealed the secretion of significantly increased amounts of IL-1ß, TNF-α, CCL-2, −3, −4, and lesser levels of IL-6, CCL-7 and −22 in macrophages exposed to 25 μg/cm 2 (a concentration representative of occupational exposure [18]) of all but Fe 3 O 4 NP as compared to unexposed cells ( Figure 6). Moreover, CXCL-8 production was also significantly higher in macrophages exposed to all but Fe 3 O 4 NP at the dose of 5 μg/cm 2 , and to all NP when used at 25 μg/cm 2 ( Figure 6). No modification of the secretion of IL-4, −5, −10, −12, −13, PDGF-AA, PDGF-BB, or TGF-ß was detected, whatever the NP utilized (data not shown). MMP-1 and MMP-7 levels in cell culture supernatants were significantly increased in MnFe 2 O 4 -treated macrophages as compared to unexposed cells (25 μg/cm 2 , Additional file 1: Figure S1). Moreover, exposure to Fe 2 O 3 NP at 25 μg/cm 2 induced an increased production of MMP-7 and TIMP-2 as compared to unexposed cells (Additional file 1: Figure S1). Finally, no modification of the expression of MMP-2, −9, −10, TIMP-1, −3, and −4 was observed irrespective of NP exposure (data not shown).

Since welding fumes contain a mixture of NP, we next questioned whether a mix of the most representative NP could have a synergistic effect on the macrophage secretome. These experiments showed that exposure of macrophages to the (1:1:1:1) combination of Fe 2 O 3 , Fe 3 O 4 , MnFe 2 O 4 and CrOOH NP didn't produce any synergistic effect in terms of cytokine or MMP/TIMP secretion ( Figure 6 and Additional file 1: Figure S1). Finally, as the majority of the welders were smokers, we analyzed if the combined exposure to NP and cigarette smoke could have a synergistic pro-inflammatory effects. These exposures did not demonstrate any synergistic effect of cigarette smoke in macrophages response to NP exposure (Additional file 2: Figure S2 and Additional file 3: Figure S3).

Effect of macrophage secretome on macrophage migration

Following the identification of the increased number of macrophages in welders lung, the consequence of an autocrine effect of macrophage secretome on the migration capacity of these cells was investigated. Concurrently with our previous data, the secretome obtained from all exposures with the exception of the Fe 3 O 4 NP-exposed macrophages was able to activate macrophage migration (Additional file 4: Figure S4).

Expression of pro-inflammatory cytokines in lung tissue sections

To further scrutinize the clinical relevance of the macrophage secretome induction by NP exposure, a series of immuno-histochemistry experiments were performed in lung tissue sections from control and welders, for a subset of cytokines that were highly produced by NPexposed macrophages. As shown in Figure 7 and quantified in Table 4, the expression of IL-1ß, TNF-α and CCL-3 was significantly higher in lung sections from welders as compared to controls, whereas no difference was observed for CCL-2 expression. For the 3 positive cytokines, and in accordance with NP tissue localization, the staining could be observed in macrophages located in the alveolar lumen as well as in fibrous regions.

Effect of macrophage secretome on human lung pulmonary fibroblast activation

Since lung from welders contained an increased number of macrophages not only in alveolar lumen but also in areas of fibrosis, and since NP induced an inflammatory secretome in macrophages in vitro that can be similarly detected in situ in macrophages localized in the same areas, we next investigated the effects of macrophage secretome on fibroblasts to myofibroblasts differentiation and fibroblasts proliferation. Since, as previously mentioned, the majority of the welders were smokers, these experiments were performed in lung fibroblasts from both non smokers and smokers, to address the specific role of cigarette smoke exposure as well as that of NP. Exposure of lung fibroblasts to the secretome of NPexposed macrophages revealed neither a modification in their α-smooth muscle actin (α-SMA) staining (Additional file 5: Figure S5A, B and data not shown), nor a change in their proliferation rate over 48 hours (Additional file 5: Figure S5C and data not shown) irrespective of the individual being a smoker or not. Moreover, no modification of the secretion of TGF-ß by fibroblasts exposed to macrophage secretome was observed (data not shown).

Discussion

In this study a combination of imaging and material science techniques were utilized to comprehensively scrutinize lung tissue samples, and for the first time thoroughly characterize welding-related NP in situ in lung tissue from welders. Moreover, given the tissue localization and the effects of such NP on macrophages in vitro, the data here strongly suggests that these NP could be responsible at least in part for the pulmonary inflammation observed in welders.

To the best of our knowledge, only a very limited number of studies has been performed in welders, identifying the presence of nanoparticles [19][20][21][22]. However, although using the valuable combined (S)TEM/EDX analysis as developed by Abraham and colleagues [23][24][25][26], these studies present some major limitations, such as a small number of patient(s) [19,21], or a very specific exposure (aluminum factory, not representative of the general occupational exposure of welders) [20]. Our study in the other hand is the first to provide persuasive evidence not only of the presence of NP in situ in human lung tissue samples, but also the identification of their chemical composition. As expected given the occupational exposure of the patients, iron was the major element present in excess in the arc welders evaluated in our study. Indeed, if the exact chemical composition of welding fumes is dependent on the material being welded and the electrode, in manual arc welding fumes and in accordance with our findings, the elemental composition is predominantly iron present as metal oxide NP of the form (M, Fe) 3 O 4 , where M may be substituted for Mn and Cr essentially [9,[27][28][29][30]. These results led us to chemically synthesize 4 different NP, representative of those found in the welders lungs; 2 speciation of pure iron oxide (Fe 2 O 3 and Fe 3 O 4 ), a mixed Mn/Fe oxide (MnFe 2 O 4 ), and an oxyhydroxide CrOOH NP. We chose to generate both Fe 2 O 3 and Fe 3 O 4 because, although our data didn't provide any evidence of the exact speciation of these iron oxides, such species have been largely described in the literature regarding welding fume chemical composition [9,27,30]. Moreover, given the observed co-localization of Mn and Fe signals in our lung samples as well as data from the literature [31], we chose to generate MnFe 2 O 4 NP as a mix metal oxide relevant to welding fume exposure. Finally, we chose to generate CrOOH oxyhydroid NP that present the same + III oxidation state and particle size in the same range as the other NP. Indeed, FeCr2O4 particles can be synthesized but not at the nanoscale, making them of no interest for our study.

We demonstrated that exposure of macrophages to the synthesized representative NP results in the production of a pro-inflammatory secretome, by itself able to further activate the migration of macrophages in vitro. Although we did not conduct experiments to identify the specific protein(s) responsible for the activation of macrophage migration, several candidates present in macrophage pro-inflammatory secretome can be proposed; CCL-2, CCL-3, CXCL-8, as they directly act as efficient monocyte, lymphocyte and neutrophil chemoattractant [32][33][34]. Moreover, it is important to underline that mediators such as TNF-α and IL-1ß, highly secreted by macrophages in response to NP (Figure 4), could also participate in the perpetuation of inflammation initiated by NP exposure since they have been shown to further induce the expression of chemotactic cytokines such as CCL-2, −3 and CXCL-2, thus sustaining the pulmonary increases of neutrophils, lymphocytes, and macrophages [32,35,36]. Similar events have been described in BAL fluid from animals exposed to welding fumes or other dusts [5,13,[37][38][39][40][41][42]. These events could happen in welders, given that we were able to detect some of these cytokines in lung tissue sections of the patients analyzed in our study. Not all NP produced the same effects in vitro, although utilized at the same mass concentration; Fe 3 O 4 was overall the less reactive NP, particularly as compared to Fe 2 O 3 , which underlines the importance of NP speciation in their individual effects. Such differences in NP effects could be related to different degrees of solubility. However, observation by TEM of 3 months old NP suspensions revealed that all nanoparticles appear similar to those observed in the initial suspension, except for MnFe 2 O 4 nanoparticles that appear slightly amorphous at their surface (data not shown). This most probably indicates a very small or no solubility at all of the nanoparticle of interest, as we should have observed a diminution of the nanoparticle diameter in case of solubility. This is also in accordance with data from literature demonstrating that iron and chromium oxides are very poorly soluble in water [43][44][45][46][47]. Interestingly, we did not observe any synergistic effect between NP when used as a 1:1:1:1 mix, suggesting a common biological mechanism of action. An LPS contamination of our NP could have been responsible for the M1-like phenotype of THP-1 macrophages in vitro [48]. However, the endotoxin content of the NP solutions was evaluated and we didn't measure any detectable levels of endotoxin, thus ruling out endotoxin contamination (data not shown). Altogether, our data strongly suggests a role for the NP identified in lung tissue samples from welders in the development of their pulmonary inflammation. This could have great implications in other populations of workers occupationally exposed to NP. As stated previously, the majority of the patients studied here were either current or former smokers which could have been troublesome during the interpretation of the data. Indeed, the presence of NP has been described in cigarette smoke [49]. Therefore, the welding origin of the NP found in welders lungs could be questioned. However, only Al and Si NP were observed in control patients, irrespective of their smoking status, thus ruling out a potential misinterpretation of the findings related to cigarette smoke NP. Moreover, we did not observe any synergistic effect between cigarette smoke and metallic NP exposure in vitro. Although we observed only a limited effect of cigarette smoke exposure alone, this underlines the unique effect of welding-related NP.

NP-loaded macrophages were present not only in the alveolar lumen but also in the fibrous tissue of lung tissue sections from welders. In chronic repetitive injury situations leading to fibrosis development (such as welding fume exposure in an occupational context), it is known that inflammation invariably precedes fibrosis [50]. Since we detected a pro-inflammatory secretome in macrophages exposed to NP, we hypothesized that this secretome could induce fibroblast differentiation into myofibroblasts. Indeed, myofibroblasts represent the major cellular effector of fibrogenic reactions, and fibroblasts have long been considered as the only cell type of origin for myofiboblasts [50]. However, the data here did not indicate any myofibroblastic differentiation. These findings could be explained by the absence of TGF-ß production in NP-exposed macrophage supernatant, as TGF-ß is the main cytokine involved in fibroblasts differentiation [50]. Moreover, one can't rule out the influence of kinetic aspects of our experimental set-up, where macrophages were exposed for 48 hours, leading to a M1-like pro-inflammatory phenotype, without the possibility of developing a later M2 phenotype, where macrophages could act as the primary effectors of later stages of repair and/or later proliferative and remodeling phases. Beside fibroblasts, fibrocytes and epithelial cells have also been described as sources of myofibroblasts [50,51]. Their response to supernatant from NP-exposed macrophages could have been interesting to evaluate given the presence of specific cytokines such as CCL-2 and Figure 6 Pro-inflammatory secretome of THP-1 macrophage exposed for 24 hours to welding-representative NP. THP-1 were exposed to 5 or 25 μg/cm 2 NP for 24 hours. Mix is a 1:1:1:1 mixture of the four NP. Macrophage pro-inflammatory secretome was assayed by Luminex. Open bars: Control. Grey bars: 5 μg/cm 2 . Black bars: 25 μg/cm 2 . N = 6 per condition. *: p < 0.05 vs Control condition.

CCL-3. Indeed, CCL-2 is a chemoattractant for fibrocytes [52], and CCL-3 has been involved in bleomycin-induced recruitment of bone marrow-derived macrophages and fibrocytes, and the subsequent development of pulmonary fibrosis [53]. This should deserve further studies.

Conclusion

In this study, thanks to the extensive physico-chemical characterization of lung tissue sample from welders, we evidenced the presence of welding-related NP in situ. Moreover, our data strongly suggest that these NP could participate in the pulmonary alterations observed in welders. These findings provide the first evidence of a link between human exposure to NP and long-term pulmonary effects.

Methods

Please refer to online data supplement (Additional file 6) for additional details.

Patients

Non-tumoral lung tissue sample from 21 arc-welders and matched 21 control patients benefiting surgical resection for primary non-small-cell lung cancer were removed and utilized in the study. All patients were subjected to a thorough occupational questionnaire. All welders had preponderantly arc-welding activities during their whole career, one third of them also performed torch and/or braze welding. The subjects essentially used Tungsten Inert Gas (TIG), Metal Inert Gas (MIG), Metal Active Gas (MAG), and/or Shielded Metal Arc (SMAW), Metal Manual Arc Welding (MMA) techniques. They welded mainly mild steel and/or stainless steel metal pieces, essentially in the following domains of activities; shipbuilding (53%), automotive industry (14%), and sheet metal work (24%). The study was approved by our institutional review board for human studies.

Histological and immunohistochemical analysis

Paraffin-embedded lung tissue sections (5 μm) were stained with hematoxylin-eosin-saffron (HES) or Perls prussian blue. The localization and severity of fibrosis was evaluated via a semi-quantitative score adapted from Roggli [54]. The presence of other respiratory lesions as well as siderophages and ferruginous bodies were quantified. Mineralogical analysis and quantification of asbestos exposure were performed as previously described [55]. Additionally, immunohistochemical (IHCh) analyses were performed on the deparaffinized tissue sections.

Characterization of pulmonary NP content

Elemental mapping in lung tissue sections X-ray microfluorescence (μ-XRF) was used to determine the elemental mapping in lung tissue sections. These experiments were performed at the European Synchrotron Radiation Facility (Grenoble, France), on ID21 and ID13 beamlines, at 7.2 and 12.5 keV edges respectively.

NP identification in lung tissue homogenates

Lung tissue homogenates were observed using a FEI Tecnai F20 STEM at 200 kV, equipped with an EDX spectrometer. Each value is given as a mean semi-quantitative score a ± SD. ns: not significant; SD: standard deviation.   Table 4.

NP identification in lung tissue sections

Paraffin-embedded lung tissue sections were deparaffined and further embedded in Epon resin. Thin sections (300 nm) were stained with Azur II dye and observed via optical microscopy. In addition ultrathin sections (70 nm) counterstained with lead citrate and uranyl acetate were observed using a JEOL1400-TEM at 120 kV, or a FEI Tecnai F20-STEM at 200 kV, equipped with EDX spectrometer for elemental analysis of the identified NP.

Synthesis of representative NP

Exposure of human macrophages to NP Cell culture

Human monocytic THP-1 cells were cultured as previously described [56], and were exposed to 1-25 μg/cm 2 NP suspensions for 24 hours (non-cytotoxic doses, data not shown). After exposure, the cell supernatant was recovered and centrifuged at 10 000 g for 15 minutes to eliminate any NP contamination.

Luminex analysis

Macrophage migration

Migration of THP-1 cells in response to supernatant from NP-exposed macrophages was evaluated using a 24-wells Boyden chamber [57].

Exposure of human fibroblasts to macrophage secretome

Human primary lung fibroblasts were obtained and cultured from a series of control subjects described in [58]. These cells were exposed to NP-exposed macrophages secretome for 48 hours. Fibroblasts expression of α-smooth muscle actin (α-SMA) was then quantified, as well as their proliferation rate (Hoechst assay).

Statistical analyses

Comparisons between controls and welders were performed using Chi squared, Fisher's exact or Mann-Whitney nonparametric tests as appropriate (GraphPad Prism software, USA). A p < 0.05 was considered as statistically significant.

Additional files

Additional file 1: Figure S1. MMP/TIMP levels in supernatant from THP-1 macrophages exposed for 24 hours to welding-representative NP. THP-1 were exposed to 5 or 25 μg/cm² NP for 24 hours. Mix is a 1:1:1:1 mixture of the four NP. Macrophage pro-inflammatory secretome was assayed by Luminex. Open bars: Control. Grey bars: 5 μg/cm². Black bars: 25 μg/cm². N = 6 per condition. *: p < 0.05 vs Control condition.

Additional file 2: Figure S2. Pro-inflammatory secretome of THP-1 macrophages exposed for 24 hours to welding-representative NP and cigarette smoke. THP-1 cells were exposed to 25 μg/cm² NP for 24 hours in presence or in absence of 5% cigarette smoke extract (CSE). Mix is a 1:1:1:1 mixture of the four NP. Macrophage pro-inflammatory secretome was assayed by Luminex. Black bars: without CSE. Dashed bars: with CSE. N = 6 per condition. *: p < 0.05 vs respective Control condition.

Additional file 3: Figure S3. MMP/TIMP levels in supernatant from THP-1 macrophages exposed for 24 hours to welding-representative NP and cigarette smoke. THP-1 cells were exposed to 25 μg/cm² NP for 24 hours in presence or in absence of 5% cigarette smoke extract (CSE). Mix is a 1:1:1:1 mixture of the four NP. Macrophage pro-inflammatory secretome was assayed by Luminex. Black bars: without CSE. Dashed bars: with CSE. N = 6 per condition. *: p < 0.05 vs respective Control condition.

Additional file 4: Figure S4. Quantification of macrophage migration after exposure to NP-exposed macrophage secretome. THP-1 macrophages were exposed to NP-exposed macrophage secretome in Boyden chamber. For each condition, the number of cells that have migrated to the bottom of the membrane was counted in eight fields. Mix is a 1:1:1:1 mixture of the four NP. N = 6 per condition. p < 0.05 vs Control condition.

Additional file 5: Figure S5. Characterization of human primary lung fibroblasts response to NP-exposed macrophage secretome. A: Representative images of immunofluorescent staining for α-SMA expression in human primary lung fibroblasts in response to NP-exposed macrophage secretome. Scale bar: 50 μm. TGF-ß was used as a positive control. B: Quantification of α-SMA expression. N = 6 per condition. C: Quantification of human primary lung fibroblasts proliferation in response to NP-exposed macrophage secretome. N = 6 per condition.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions PA, ASD, JB and SL designed the study. BC performed the face-to-face interview, PA and JCP selected the patients, and PA and FGS performed the histological analysis. MABG performed the mineralogic analyses, and ASD all biological experiments. ASD, BF, JD and SL performed the μXRF experiments, and ASD performed all other imaging experiments. GB and OD synthesized and characterized the NP. PA, ASD and SL drafted the manuscript, and SL and JB were the main additional participants to its further elaboration. All authors read and approved the final manuscript.

Figure 1
1Histological observation of lung tissue sections. Representative optical microscopy images of lung tissue sections from a Control and a Welder. Serial sections were stained with hematoxylin-eosin-saffron (HES), CD68 or Perls. Scale bar: 100 μm. Inset: higher magnification of the welder sample.

Figure 2
2Elemental mapping of lung tissue sections. A: Representative elemental map obtained by X-ray micro-fluorescence (μXRF) in a Control and a Welder. Signal of sulphur: green. Signal of iron: blue. Signal of manganese: red. Scale bar: 100 μm. For each element, fluorescence intensities are proportional to the total amount of element. B: Quantification of the signal obtained for each element, given as a ratio to that of sulphur. *: p < 0.05 vs Control, **: p <0.01 vs Control, n = 6-8 per group. Open circles: Controls (grey horizontal bar: mean value). Black squares: Welders (black horizontal bar: mean value).

Figure 3
3Identification of NP in lung tissue homogenates. A: Typical STEM images obtained in sodium hypochlorite-digested lung tissue samples from welders. Scale bar: 50 nm. B: EDX spectra of individual NP detected by STEM.

Figure 4
4Identification of NP in lung tissue sections. A: Representative optical microscopy image of a thin section (Azur II staining) of a welder. Inset: higher magnification of a macrophage-containing fibrous region, observed by TEM. B: Representative EDX spectrum of individual NP detected by TEM.

Figure 5
5Characterization of welding-representative NP. A-D: Representative TEM images of Fe 2 O 3 (A), Fe 3 O 4 (B), MnFe 2 O 4 (C) and CrOOH (D) NP. E-H: Corresponding XRD spectra obtained for each NP. The three important 2θ values (arrow) of XRD spectra were use to identified synthesized NP as Fe 2 O 3 (E), Fe 3 O 4 (F), MnFe 2 O 4 (G) and CrOOH (H).

a
Semi-quantitative score [0: 0 positive cells/slide; 1: 1-5 positive cells/slide; 2: 5-10 positive cells/slide; 3: 10-50 positive cells/slide; 4: >50 positive cells/slide].

Figure 7
7Expression of IL-1β, CCL-2, CCL-3 and TNF-α in lung tissue sections. Representative images obtained after immunostaining for IL-1ß, CCL-2, CCL-3 and TNF-α in lung tissue sections from a Control and a Welder. Scale bar: 50 μm. Quantification of the immunostaining is given in

Fe 2 O 3 ,
3Fe 3 O 4 , MnFe 2 O 4 and CrOOH NP were synthesized in aqueous solution using the micro-waved-assisted sol-gel method. Particle nature was determined by X-Ray diffraction (XRD), and their size and shape were evaluated using the Debye-Scherrer equation on several diffraction peaks, together with an analysis of TEM images.

Table 1
1Clinicopathological characteristics of control and welder patients.Controls
Welders P value
(n = 21)
(n = 21)

Table 2
2Histological analysis of lung tissue in control and welder patientsControls
Welders P value
(n = 21)
(n = 21)

Siderophages

Presence (n patients)
10
17
0.024

Number (Mean semi-quantitative
score ± SD) b

0.71 ± 0.90 1.57 ± 1.17 0.019

Ferruginous bodies (n patients)

Presence (n patients)
6
16
0.002

Number (Mean semi-quantitative
score ± SD) b

0.43 ± 0.81 1.81 ± 1.57 0.002

Fibrotic lesions
(Number of patients)

Peribronchiolar
16
20
ns

Perivascular
14
20
0.018

Paraseptal
8
10
ns

Alveolar
3
5
ns

Diffuse interstitial
6
5
ns

Pleural
4
2
ns

Severity of fibrosis a

(Mean semi-quantitative score ± SD)

Global
2.29 ± 1.59 3.91 ± 1.51 0.003

Peribronchiolar
1.10 ± 0.77 1.86 ± 0.85 0.005

Perivascular
1.05 ± 0.92 1.81 ± 0.81 0.010

Alveolar and peribronchiolar
1.57 ± 1.12 2.67 ± 1.16 0.004

Other respiratory lesions

Bronchiolectasis
16
13
ns

Bronchiolar metaplasia
2
0
ns

Bronchiolar dysplasia
1
5
ns

Respiratory Bronchiolitis -Intertitial
Lung Disease

10
10
ns

Desquamative intertitial pneumonitis
12
9
ns

Endogenous Lipoid Pneumonitis
1
3
ns

ns: not significant; SD: standard deviation.

a

Semi-quantitative score [4 classes = 0: absent; 1: rare; 2: frequent; 3:
important]. Adapted from Roggli classification [54].

b

Semi-quantitative score [5 classes = 0: 0/slide; 1: < 5/slide; 2: 5 to 10/slide; 3:
10 to 50/slide ; 4: > 50/slide].

Table 3
3Mineralogical analysis and asbestos exposure quantification of lung tissue homogenates in control and welder patientsControls
Welders
P value
(n = 21)
(n = 21)

Table 4
4Semi-quantitative score of cytokine expression in lung tissuesControls
Welders
P value
(n = 7)
(n = 10)

IL-1β
1.71 ± 1.11
3.60 ± 0.52
0.002

TNF-α
0.83 ± 0.75
3.43 ± 1.81
0.042

CCL2
1.86 ± 1.57
2.00 ± 1.31
ns

CCL3
1.29 ± 1.25
3.00 ± 1.00
0.015

http://www.particleandfibretoxicology.com/content/11/1/23
AcknowledgmentsThe authors would like to thank Laurent Martinon and Xavier Janson (Laboratoire d'Étude des Particules Inhalées), Soizick Chamming's (Institut Interuniversitaire de Médecine du Travail de Paris -Ile de France) for their participation in the study, Dr Blandine Briquel-Jean-Luc for her helpful contribution to collect complete work history data of each subject, and Ali Kermanizadeh for the English editing. This work has benefited from the facilities and expertise of the Imagif Cell Biology Unit of the Gif campus (www.imagif.cnrs.fr), which is supported by the Conseil Général de l'Essonne, and of the electronic microscopy plateform of Institut de Chimie des Matériaux Paris-Est (ICMPE, UMR 7182, Thiais, France).Submit your next manuscript to BioMed Central and take full advantage of:
Angélique Simon-Deckers and Grégory Beaune were both fellows from ANR. Jorge Boczkowski and Sophie Lanone were both recipients of a Contrat de Recherche Translationnelle, between Inserm and CHU Mondor (JB) and Inserm and CHI Créteil (SL). This work was supported by funds from INSERM. Chancellerie des Universités de Paris (Legs POIX), Université Paris Est-Créteil ; Département Hospitalo-Universitaire Ageing-Thorax-Vessel BloodThis work received the support of Labex SERENADE 11-LABX-0064 and form DHU A-TVBThis work was supported by funds from INSERM, Chancellerie des Universités de Paris (Legs POIX), Université Paris Est-Créteil, Agence Nationale de la Recherche (grant ANR-09-CESA-017), Fond de Dotation -Recherche en Santé Respiratoire (SPLF) and C'Nano (grant AAP09-NanoSoud). Angélique Simon-Deckers and Grégory Beaune were both fellows from ANR. Jorge Boczkowski and Sophie Lanone were both recipients of a Contrat de Recherche Translationnelle, between Inserm and CHU Mondor (JB) and Inserm and CHI Créteil (SL). This work received the support of Labex SERENADE 11-LABX-0064 and form DHU A-TVB (Département Hospitalo-Universitaire Ageing-Thorax-Vessel Blood).

. France Créteil, 94000Université Paris Est-Créteil, Faculté de MédecineCréteil, France. 3 Université Paris Est-Créteil, Faculté de Médecine, 94000

Laboratoire de Physique des Solides. France Créteil, CNRS UMR. 8502491400Créteil, France. 4 Laboratoire de Physique des Solides, CNRS UMR 8502, 91400

. France Orsay, Chu Caen, Caen, FranceService d'Anatomo-PathologieOrsay, France. 5 CHU Caen, Service d'Anatomo-Pathologie, 14000 Caen, France.

. U1086 Inserm, Faculté De Médecine, Caen, France; Paris, France. 8 CNRS; Paris, FranceSorbonne Universités, UPMC Univ. Paris 06, UMR 7574, Laboratoire de Chimie de la Matière Condensée de Paris ; UMR 7574, Laboratoire de Chimie de la Matière Condensée de ParisINSERM, U1086, Faculté de Médecine, 14000 Caen, France. 7 Sorbonne Universités, UPMC Univ. Paris 06, UMR 7574, Laboratoire de Chimie de la Matière Condensée de Paris, F-75005 Paris, France. 8 CNRS, UMR 7574, Laboratoire de Chimie de la Matière Condensée de Paris, F-75005 Paris, France.

. France Collège De, 12 CHU Henri Mondor75013Paris, France. 10 CHU Caen; Caen, France; Paris, France; FranceUMR 7574, Laboratoire de Chimie de la Matière Condensée de Paris ; Service de Santé au Travail et Pathologie Professionnelle11 Laboratoire d'Etude des Particules InhaléesService d'explorations fonctionnelles respiratoires, 94000 CréteilCollège de France, UMR 7574, Laboratoire de Chimie de la Matière Condensée de Paris, F-75005 Paris, France. 10 CHU Caen, Service de Santé au Travail et Pathologie Professionnelle, 14000 Caen, France. 11 Laboratoire d'Etude des Particules Inhalées, 75013 Paris, France. 12 CHU Henri Mondor, Service d'explorations fonctionnelles respiratoires, 94000 Créteil, France.

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Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders. Andujar, Particle and Fibre Toxicology. 1123Cite this article asCite this article as: Andujar et al.: Role of metal oxide nanoparticles in histopathological changes observed in the lung of welders. Particle and Fibre Toxicology 2014 11:23.


Unjustifi ed Restrictions on Letters to the Editor

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Unjustifi ed Restrictions on Letters to the Editor
88348F5474829E92D5C4BC5169DE2A7B10.1371/journal.pmed.0020126
Editors of medical journals accept that published research should be open to comment and correction in published correspondence ([1]; Box 1)."Post-publication peer review" enables comments on, clarifi cations of, and corrections to published research.All journals should have a correspondence page for this purpose.I previously criticised the effective "statute of limitations" in several leading general medical journals "whereby authors of papers are immune to disclosure of methodological weaknesses once some arbitrary (short) period has elapsed" [2].Such a time limit discourages post-publication peer review, with potential correspondents deterred by the short and unambiguous deadline.I suggested that journals with such a policy should reconsider.The word limit on that article precluded additional adverse comments on journals' word limits for letters, although they were presented in Table2of that article [2].Subsequently, three of the six journals did revise their instructions [3-5], but each imposed tougher restrictions on letters, reducing either the maximum time limit, the maximum length, or both.The strictest current requirements are a two-week limit by The Lancet and a 175-word limit by the New England Journal of Medicine.Editors are seemingly falling over themselves to speed up and shorten letters, but this behaviour is inappropriate for a scientifi c journal.The key characteristic of science is not its infallibility, a quality it clearly does not and cannot have, but its self-correcting ability.The decision by medical editors to stifl e debate is misguided [2,6].A time limit, especially a very short one, signals that speed is more important than content, that convenience takes precedence over science.While it is reasonable to encourage early comments, there should be no time limit on comments aimed at clarifying or criticising study methodology.Likewise, it will often be impossible to explain the subtleties of methodological problems in 400 words, and impossible in only 175.Additional restrictions on the number of authors and references are also questionable.I am disappointed that PLoS Medicine has imposed a time limit of four weeks on correspondence.As explained above, I believe that such a limit is mistaken.The word limit of 750 words is generous by comparison to established general medical journals, but even this should be open to fl exibility should the circumstances merit it.In this world of Web-based journals and Web pages for print journals there is no real cost to permitting longer and later letters on the web while keeping the print version timely and terse.

Box 1. "Editors Should Promote Self-Correction in Science and Participate in Efforts to Improve the Practice of Scientifi c Investigation by:

• "Publishing corrections, retractions, and letters critical of articles published in their own journal.

• "Playing an active role in investigating and preventing fraud.

• "Taking responsibility for improving the level of scientifi c investigation and medical writing in the larger community of potential authors.

• "Giving authors an opportunity to review and approve edited manuscripts before they are published.

• "Participating in efforts to detect and prevent publication bias-for example, by collaborating with registries of controlled trials and publishing protocols."

Association between Injections and HIV Incidence Naveed Zafar Janjua, Khabir Ahmad, Arshad Altaf, Mohammad Imran Khan, Hasan Bin Hamza

The article by Lopman and colleagues [1] has created more controversy than it's cured.Their conclusion that unsafe injections "do not play a major role in the transmission of HIV in rural Zimbabwe" cannot be based on the data they have presented.There are many major problems with their conclusions.

First, they did not assess at all whether injections received by the participants in the study were safe or unsafe.All they assessed was whether participants had received an injection or had been pricked by a needle during the past three years.The possibility that most of the injections in the setting were "safe" cannot be ruled out, and the data presented did not in any way address the issue of an association between "unsafe injection" practices and HIV infection.

Second, the recall period used by the investigators was too long.Cognizant of this problem, the World Health Organization has been proposing a three-month recall period for assessing frequency of injections.Although this recall period is 12 times shorter than the one used by the authors, we found in our studies in Pakistan that, even then, people had great diffi culties recalling injections.It must be mentioned that injections are very frequent procedures in Pakistan [2].

Third, it seems that aside from injections, the investigators have included only data from sexual histories in their study; hence, other potential sources of exposure such as minor and major surgical procedures, dental instrumentation, and tattooing or other traditional practices involving scarifi cation have been missed.

Fourth, it is not clear how "needle prick" was defi ned.Solid needles are also considered needles, but injury caused by these is less likely to transmit HIV than that caused by hollow bore needles.

Fifth, the authors have failed to quantify exposure.The risk of contracting HIV increases as the number of unsafe injections increases [3].The incidence of disease among people who received one injection during follow-up compared with those who received 20 injections would clearly be different.This relationship has been clearly seen in the case of hepatitis C infection [4].

Sixth, the authors recommend that policymakers "should concentrate more on trying to prevent infection from unsafe sex" than on injections.But they have failed to assess whether the sex was unsafe or otherwise.

Further, we believe that even if the methodology is considered absolutely fl awless, the current conclusion can only apply to a particular population and geographic area because the proportion of disease attributable to various exposures depends on the relative distribution of exposures in the population.For example, it is argued that in India (with the world's second largest HIV/AIDS population, more than 5 million) the HIV epidemic started as a result of high-risk sexual behaviors, but the number of injections per person is high and reuse of syringes in the health-care sector is widespread (N.K. Arora, personal communication).Therefore, unsafe medical injections have the potential to propagate this epidemic.Also, injections transmit many other pathogens like hepatitis B virus and hepatitis C virus.The infections that they cause have a very high morbidity and mortality.Hence, the need and urgency of intervention to decrease the overuse of injections and improve the safety of desired injections should not be questioned.Each country should make appropriate allocation of resources according to its own needs.

Naveed Zafar Janjua (email@example.com their attempt to defend heterosexual transmission as the driving force for HIV epidemics in sub-Saharan Africa and dismiss the evidence for unsafe injections (or other percutaneous exposures) as a major source of HIV transmission [1,2], Lopman and colleagues [3] assert that "unsafe medical injections can be confi dently excluded as a major source of HIV infection" in Manicaland, Zimbabwe.Their methods and results are glaringly insuffi cient to generate the confi dence they seek.Confi dence requires thorough evaluation, which was not done in their study [3].

Although the authors quantifi ed the number of sexual partners, they failed to quantify the number of injections [3], which undermines the likelihood of detecting an effect of injections, as does adding the statistical noise of "needle pricks" (how was that question phrased?) to injections.We fi nd it diffi cult to believe that the authors were unaware of either the dose-dependency issue or the implications of using a weak measure of a vector that they are transparently invested in dismissing.In addition, the recall interval was inordinately long (up to three years).It is unreasonable to expect accurate data from subjects who are not multiply prompted to stimulate recall.

As a secondary issue, their adjustment of the risk ratio associating HIV with injections against age is problematic.Adjusting a causal variable against age (which is a proxy for causal variables) may reduce the true association.Their Table 1 shows that injections in women vary by age in the same pattern that HIV incidence varies by age.It might be that when they adjust for age, the risk ratios for age capture the association, leaving a diminished association for injections.

Confi dence in their report is also undermined by their approach to their own fi nding that sexual behavior was unrelated to risk of incident HIV.Indeed, using the same standard of evidence that the authors applied to their null medical-injection fi nding, the abstract, discussion, and press release information should also have proclaimed that sex was "confi dently excluded" as a risk for HIV.Indeed, this would be consistent with the largest studies of HIV risk in Africa, including one in their own backyard (Manicaland) [4], which found little association of measured sexual behaviors with HIV risk.It would also be consistent with the many intervention studies reporting no benefi t from condompromotion programs [5], as well as with observation of the opposite trajectories of the HIV and STD epidemics observed in Zimbabwe [6].

Of additional concern is Lopman et al.'s fi nding that 13 of 67 individuals who seroconverted reported no sexual partners in the long inter-survey period.Indeed, their Table 2 data show that women with no reported sexual partners have higher HIV incidence than women who report any partner during the three-year interval (1.56 HIV cases per 100 personyears [12/770] for the former compared to 1.21 [36/2975] for the latter).They reveal their a priori conviction by forcing this datum into the procrustean sexual bed, inferring that it is explainable by unreported sexual activity rather than unreported or unmeasured percutaneous exposure.The authors blame underreporting of sexual behavior without using techniques shown to dramatically improve valid reporting [7].

Despite this remarkable lack of association in their women respondents between HIV incidence and number of sexual partners, they counterintuitively suggest (in the patient summary) that the important issue is sexual transmission.It seems to us that if sex doesn't appear to explain high HIV incidence, then one should recommend looking for what does, which the authors do not do.Rather than relying on the case-control approach, what is needed is intensive contact tracing (with viral sequencing of HIV specimens from index cases and their infected contacts to elucidate transmission relationships [8]) and a rigorous inventory of possible exposures and vectors [7].The contact tracing and conscientious environmental risk probing of recent public-health responses to avian infl uenza and severe acute respiratory syndrome are an example of a useful and superior step to the case-control approach.

In brief, what is truly driving HIV transmission in sub-Saharan Africa will not be resolved by hastily implemented weak variables or by dismissive comments about Africans' self-reports of sexual behavior, especially comments unencumbered by data.Africans, science, and public health deserve better research [7].

Authors' Reply: Don't Let the Hypothesis Slip

In 2003, Brody and colleagues called for researchers to publish analyses investigating the hypothesised importance of medical injections in the transmission of HIV in Africa [1,2].Considering the general failure of HIV/AIDS control programs and the neglect of this subject, we believed they were right to raise this controversial hypothesis, so we added a question to our fi eld survey and performed a fresh analysis to test the strength of association between injections and HIV incidence [3].Therefore, we are disappointed that Brody and Potterat think that we are "transparently invested in dismissing" the hypothesis [2].Now that pertinent incidence data (where the timing of exposure and event can be determined) have been published for Manicaland, Zimbabwe, and Rakai, Uganda, and have shown a lack of association with injections, we think it is unfair to belittle the diffi culty of collecting data and to claim that we have not gone to great lengths to collect high-quality data on sexual behaviour [3,4].On the contrary, because of the general problems in generating reliable responses to questions about sexual behaviour [5,6], the Manicaland HIV/ STD Prevention study has pioneered the use of informal, confi dential voting interview methods [6].Brody and Potterat state that we found "that sexual behaviour was unrelated to risk of incident HIV".However, in women, having a history of STD symptoms, having multiple sexual partners, or being widowed/separated/divorced (a proxy that a previous sexual partner died of HIV) were associated with HIV incidence.In men, the associations of HIV and sexual behaviour did not reach statistical signifi cance because of the small number of seroconversions.

It is true that women with one reported sex partner did not have a higher incidence than women with no reported partners.However, Brody and Potterat fail to point out that women with multiple sex partners had the highest incidence (31.3 cases per 1,000 person-years) and that rates were lower in men with no sex partners (3.1) than in those with one sex partner (13.6) or multiple sex partners (14.9).These analyses were performed on only a subset of the Manicaland cohort, but other publications have demonstrated the role of sexual behaviours as risk factors for HIV in this population [7].

We agree that our measure of injections was not perfect, and Brody and Potterat reiterate many of the limitations discussed in our paper: we used a binary (yes/no) measure of exposure, which did not capture the number of injections and had a fairly long follow-up period of three years.These dimensions are being measured in the next round of the cohort study.In the published data, it is possible that some cases had their exposure misclassifi ed, but as many as 40 (60%) of the individuals who seroconverted reported not to have received an injection.Post hoc power calculations (Figure 1) demonstrate that if there was a risk of 2.27 associated with injections, the fi nding would have been statistically signifi cant.The crude rate ratio for both sexes was 1.1 (95%; confi dence interval: 0.7-1.8),which is not evidence that injections are a major transmission route of HIV.

Brody and Potterat also claim that our statistics are fl awed because we controlled for age in the analysis.This is a moot point.We presented both univariable and age-adjusted rate ratios of injection exposure-neither showed an association.

We fi nd it strange that Brody and Potterat reference themselves for a study performed in our "own backyard", which was actually the baseline survey for our current study, and then mislead by saying that it shows little association between sexual behaviour and HIV risk.Lifetime number of sexual partners was in fact a very strong determinant of HIV status in this population [6,7].

In their separate response, Naveed Zafar Janjua and colleagues point out a number of important aspects concerning injection epidemiology and health careassociated infections [8].First, there is, by defi nition, a difference between safe and unsafe injections.Second, heightened risks may be associated with "minor and major surgical procedures, dental instrumentation, and tattooing or other traditional practices involving scarifi cation".(Although not part of our original report, 16 HIV-negative individuals reported to have received a blood transfusion in the followup period.None of them seroconverted.)Third, a "needle prick" is a general term that captures lacerations with solid needles as well as those with a borehole.And fourth, the risk associated with receiving one injection is not the same as that for multiple injections, with certain types of injections carrying more risk than others.

However, these concerns expressed by Janjua et al. are not pertinent to the hypothesis that we were testing: are injections a major route of transmission of HIV in this population in Manicaland Province in Zimbabwe?This analysis was motivated by the arguments of Gisselquist et al. that injections are the main driver of HIV transmission in southern Africa [1].To be clear-we were not testing whether exposure to contaminated needles is a risk factor (clearly it is), whether certain types of injections carry more risk than others (clearly they do), or whether needles are a driver of the epidemic in certain populations in the world (clearly they are).The fact that there is no evidence of association between receipt of injections (of any number) and HIV incidence, before and after controlling for confounding variables, allows us to conclude that injections "do not play a major role in the transmission of HIV in rural Zimbabwe" [3].

The global HIV problem is not a single epidemic.In eastern Europe, over 50% of HIV infections are among users of injection drugs [9]; in Pakistan, people receive on average eight injections per year compared with about one in sub-Saharan Africa [10].Our fi ndings apply to Manicaland and may be relevant for similar epidemic patterns in southern Africa.They are not generalisable to all locations, but they do refute the hypothesis that HIV is transmitted through medical injections in the study population.Assuming power of 90% at the 95% signifi cance level [11], a rate ratio of 2.27 or greater would have achieved a signifi cant result given the 5,500 years of observation in the sample: Sample size = 2 where u = 1.28, which is the one-sided percentage point of the normal distribution corresponding to 100% minus the power; v = 1.96, which is the percentage point of the normal distribution corresponding to the two-sided signifi cance level; and µ1 and µ2 are the seroconversion rates in exposed and unexposed individuals, respectively.
(u + v) 2 (µ 1 + µ 0 ) (µ 1 -µ 0 ) 2

Fundamental Limits to the Precision of Early Warning Systems for Epidemics of Infectious Diseases

The development of early warning systems (EWSs) for epidemics of infectious diseases based on recurrent statistical patterns in other kinds of information, particularly data on climate, is an active area of research [1,2].Judging from the estimated burden of diseases for which EWSs might be developed, such systems, if effective, would contribute greatly to human welfare and could potentially save many lives [2].According to a recent report [2], EWSs have two principal aims: (i) to identify whether an epidemic will occur and (ii) to predict the number of cases that will result from it.For directly transmitted diseases, this second aim may be unattainable at the desired levels of precision, regardless of the quality of information.

As an example, in a recent report on the relationship between climate and outbreaks of meningococcal meningitis, the authors found that the timing of epidemics is highly predictable from information on the dynamics of a seasonal weather pattern, the Harmattan winds, but that the fi nal epidemic size is not [1].This fi nding is not surprising.The characteristics of disease outbreaks, particularly outbreaks of emerging diseases to which human populations are highly susceptible, prevent highly precise forecasts.

The reason that precise estimates of the fi nal epidemic size cannot be obtained can be understood intuitively.Consider the following description of a typical outbreak.Characteristically, an outbreak begins with a small number of initially infectious individuals.Subsequent infectious contacts are mediated by a wide range of social interactions-contacts within and among households and communities-so that even individuals that are virtually identical can differ considerably in the number of secondary infections they cause.This is a micro-scale cause of variation compared with macro-scale, population-level sources of variation.The important implication for EWSs is that in such situations, especially where the basic reproductive ratio of infections (R0) is initially very high but is rapidly reduced (perhaps by public-health interventions), small deviations in the realized number of infectious contacts are amplifi ed, resulting in relatively large variation in the fi nal size of the outbreak.Because this variation refl ects differences in individual behavior and not macroscopic characteristics of epidemic spread, it is unlikely that climate or other data contain any information about this source of variation (though such data do contain information about macroscopic variation).

A more formal explanation of this phenomenon can be formulated based on a simple model of an epidemic in which an EWS captures all macroscopic causes of variation in the fi nal epidemic size but no microscopic causes.Obviously, an EWS cannot realistically be expected to capture even all the macroscopic information.Thus, this limit to precision is a fundamental limit and should be interpreted as a theoretical upper bound on forecast precision.The simplest case considers a disease with only two macroscopic epidemiological characteristics, an infection rate and a removal rate, which may change over time as in the case of meningococcal meningitis.In particular, we assume that there is no immunity in the population and that infection and removal are independent in time.This model of disease dynamics belongs to a class of stochastic processes known as nonhomogenous birth-death processes, which, conveniently, turn out to be reasonably tractable.More than 50 years ago, Kendall [3] showed how models for the mean and the variance in the fi nal epidemic size are affected by these parameters.The variance can be interpreted as a measure of the precision with which the fi nal epidemic size can be predicted.Kendall's results can be broken down to show that this quantity is equal to the sum of the average fi nal epidemic size and another quantity (x) minus one.For most realistic epidemiological parameters, this other quantity, which is related to the covariance between fi nal epidemic size and the size of the infected population, will be much greater than one.In these cases, the variance in the fi nal epidemic size will be much greater than the average fi nal epidemic size itself.

This fundamental limit to the precision of forecasts does not imply that EWSs cannot be used effectively to plan a response to outbreaks.Rather, it suggests what expectations of EWSs are reasonable.Further, since the precision with which forecasts of the fi nal epidemic size can be obtained will depend on many disease-specifi c properties and maybe other factors, too, case studies of the potential effectiveness of EWSs for different diseases are needed.These studies should exploit recent advances in modeling birth-death processes [4] to gain further understanding of the differences among diseases and of the causes of geographic variation in the intensity of epidemics.Finally, notwithstanding limits to precision, the benefi ts to be obtained from estimates of the average fi nal epidemic size and the timing of epidemics alone may warrant considerable investment in EWSs.

HIV

Janjua NZ, Ahmad K, Altaf A, Khan MI, Hamza HB (2005) Association between injections and HIV incidence.PLoS Med 2(5): e139.Copyright: © 2005 Janjua et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Competing Interests: The authors have declared that no competing interests exist.DOI: 10.1371/journal.pmed.0020139

Figure 1 .
1
Figure 1.Plot of the Detectable Rate Ratio as a Function of Sample Size

John M. DrakeUniversity of California at Santa Barbara Santa Barbara, California, United States of America E-mail: firstname.lastname@example.com
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