Taché Sylviane

Corpet Van Beek Erik

Denis Corpet
email: <d.corpet@envt.fr>

Polyethylene glycol, unique among laxatives, suppresses aberrant crypt foci, by elimination of cells

Keywords: Acute Toxicity Tests, Animals, Azoxymethane, toxicity, Carcinogens, toxicity, Cathartics, pharmacology, therapeutic use, Colonic Neoplasms, chemically induced, prevention & control, Feces, chemistry, Polyethylene Glycols, pharmacology, therapeutic use, Precancerous Conditions, chemically induced, prevention & control, Random Allocation, Rats, Rats, Inbred F344 aberrant-crypt-foci, ACF, chemoprevention, colorectal cancer, laxative, polyethylene-glycol, PEG, rat, toxicity, toxicology, prevention, preneoplastic, cancer, block polymer, polycarbophil, karaya gum, psyllium, mannitol, sorbitol, lactulose, propylene glycol, magnesium hydroxide, sodium phosphate, bisacodyl, docusate, paraffin oil, TUNEL, apoptosis, fecal bulk, fecal moisture, fluorescein dextran

Background

Polyethylene glycol (PEG), an osmotic laxative, is a very potent inhibitor of colon cancer in rats. In a search for mechanisms, we tested the hypothesis that fecal bulking and moisture decreases colon carcinogenesis. We also looked for PEG effects on crypt cells in vivo.

Methods

Fischer 344 rats (N 272) were given an injection of the colon carcinogen azoxymethane. They were then randomized to a standard AIN76 = diet containing one of 19 laxative agents (5 w/w in most cases): PEG 8000 and other PEG-like compounds, carboxymethylcellulose, % polyvinylpyrrolidone, sodium polyacrylate, calcium polycarbophil, karaya gum, psyllium, mannitol, sorbitol, lactulose, propylene glycol, magnesium hydroxide, sodium phosphate, bisacodyl, docusate, and paraffin oil. Aberrant crypt foci (ACF) and fecal values were measured blindly after a 30-day treatment. Proliferation, apoptosis, and the removal of cells from crypts were studied in control and PEG-fed rats by various methods, including TUNEL and fluorescein dextran labeling. Results PEG 8000 reduced nine-fold the number of ACF in rats (p<0.001). The other PEGs and magnesium-hydroxide modestly suppressed ACF, but not the other laxatives. ACF number did not correlate with fecal weight or moisture. PEG doubled the apoptotic bodies per crypt (p<0.05), increased proliferation by 25 50 (p<0.05) and strikingly increased (>40-fold) a fecal marker of epitheliolysis in the gut -% (p<0.001). PEG normalized the percentage of fluorescein dextran labeled cells on the top of ACF (p<0.001).

Conclusions

Among laxatives, only PEG afforded potent chemoprevention. PEG protection was not due to increased fecal bulking, but likely to the elimination of cells from precancerous lesions.

Introduction

The prevention of colorectal cancer could employ chemopreventive agents, but known agents afford only modest protection. We have previously established that a diet supplemented with an osmotic laxative suppresses an early putative step in the development of colon cancer in rats ( ), namely, the number of aberrant crypt foci (ACF) induced by an azoxymethane (AOM) injection ( ). The laxative was polyethylene 1 2

glycol 8000 (PEG), whose formula is H-(O-CH -CH ) -OH, with n 200. PEG and PEG-like block-polymer pluronic F68 are the most potent = known suppressors of ACF in rats ( , ). PEG also strikingly suppresses the occurrence of AOM-induced cancers in rats ( , ). This AOM-rat 3 4 5 6

model predicts the chemopreventive efficacy of dietary agents in humans ( ). Last, a population-based study shows that PEG-based laxative 7

users have a halved risk of colorectal tumors ( ). 8

The mechanism by which PEG suppresses ACF and cancer is not known. Fecal weight and moisture are more than doubled in PEG-treated rats ( ). Constipation, by contrast, is associated with increased risk of colon cancers in several case-control studies ( ). We thus looked for 1 [START_REF] Sonnenberg | Constipation and cathartics as risk factors of colorectal cancer -a meta-analysis[END_REF][START_REF] Jacobs | Constipation, laxative use, and colon cancer among middle-aged adults[END_REF][START_REF] Roberts | Constipation, laxative use, and colon cancer in a North Carolina population[END_REF] the chemopreventive effect of several laxative agents ( ). We tested seven large bulking polymers (MW>2000), six poorly absorbed osmotic 12 agents (MW<400), a lubricating oil, two secretagogues, and three PEGs. Since many studies report the modest protective effect of wheat bran (

), and the promotion of carcinogenesis by anthranoid laxatives ( ), we did not test them. In vitro studies suggest a second possible 13 [START_REF] Vangorkom | Review article: anthranoid laxatives and their potential carcinogenic effects[END_REF][START_REF] Borrelli | Effect of bisacodyl and cascara on growth of aberrant crypt foci and malignant tumors in the rat colon[END_REF][START_REF] Mascolo | Does senna extract promote growth of aberrant crypt foci and malignant tumors in rat colon?[END_REF] mechanism: PEG halts proliferation ( ), induces apoptosis ( ), and restores differentiation ( ) of adenocarcinoma cells in culture. We thus 17 18 19 sought a PEG effect on these functions in the colon of rats. A third hypothetical mechanism is that, like mucus, PEG might protect the mucosa ( ). Indeed, cell membrane can reseal after being damaged by mechanical stress in the gut ( ). Pluronic facilitates this repair process in vitro ( 2021

), We tested this hypothesis using fluorescein dextran ( ). [START_REF] Togo | Steinhardt RA The mechanism of facilitated cell membrane resealing[END_REF] 21

Materials and Methods

General Design and Initiation of Carcinogenesis

Seven sequential experiments were conducted in rats. Five 30-day studies tested the effect of laxative agents on AOM-induced ACF.

Another 30-day study tested the effect of PEG on cell proliferation and apoptosis. Unless otherwise stated we used PEG 8000. One 100-day study made use of a fluorescent marker to see whether PEG protects or kills crypt cells. A total number of 272 four-week old Fisher 344 rats were obtained from Iffa-Credo (Lyon, France). They were acclimatized to the animal colony for one week, housed by pairs in stainless steel wire drop-bottom cages, in a temperature of 22 2 C and with 12h/12h light and dark cycles. AIN76 diet from UAR (Villemoisson, France)

± °and tap water were provided Each rat was given one AOM i.p. injection to induce ACF (20 mg/kg in saline), and randomized 7 days ad libitum. later to receive treatment. All chemicals came from Sigma (St. Quentin, France), unless stated otherwise. Body weights, food and water intake were monitored weekly. Daily fecal output was measured on freeze-dried feces collected under each cage of two rats for 24 hours. Fecal humidity was measured on fresh pellets obtained directly at the anus of each rat (weighed before and after freeze-drying). Thirty and seven days (ACF studies), or 100 130 days (fluorescent marker study) after the AOM injection, the animals were sacrificed by carbon dioxide asphyxiation. Investigator with French official agreement 31 121 handled animals, and care was in accordance with the guidelines of the # -European Council.

Studies of Laxative Agents

The effect of dietary laxative agents on AOM-induced ACF was compared to the standard AIN76 diet (control), in five 30-day studies.

They involved 32, 36 and 42 male, and 48 and 52 female rats (no gender effect was seen). In each study some rats were randomized to a control group, and to a group given dietary 5 (w/w) PEG 8000 (ICN, Orsay France). Laxative agents which were given to the other groups of rats % were: 5 of calcium polycarbophil (brought by 7 Fibercon, Whitehall-Healthcare, Madison, NJ), carboxymethylcellulose (of medium weight and moisture, fecal water osmolarity, and alkaline phosphatase (ALP) activity. A single observer scored all colons blindly for ACF, in a randomized order, by Bird s procedure ( ). Fecal water was obtained by adding distilled water to feces directly obtained from the anus of each ' 2 rat (1.5 ml per 0.15 g of freeze-dried sample), and processed as previously described ( ). Osmolarity of fecal water was measured in duplicate 1 with freezing point depression (Roebling,; Osmomat, Gonotec; Berlin, Germany). ALP is released in the fecal stream when epithelial cells are lysed ( , ). Intestinal ALP activity in fecal water was measured according to Lapre ( ), using a specific kit with p-Nitrophenyl phosphate 23 24 24 as a substrate, and L-phenylalanine as a specific inhibitor of the intestinal isoenzyme. That PEG did not modify ALP values was specifically checked in vitro.

Study of PEG effect on Cell Proliferation and Apoptosis

Sixteen male rats received an AOM injection seven days before being randomized to a control group given pure water, and a treated group given 5 PEG in the drinking water for 30 days. Sixty minutes before sacrifice each rat received an i.p. injection of 5-bromo-2 -deoxyuridine % ′

(BrdU, 50 mg/kg in NaCl 9g/l). A 1 cm piece of distal colon was processed for histology after fixation in buffered formalin [START_REF] Jacobs | Constipation, laxative use, and colon cancer among middle-aged adults[END_REF].

Study of PEG Effect on Cell Resealing and Removal in Normal and Aberrant Crypts

Twelve rats received an AOM injection, ten rats did not. They were sacrificed 106 131 days after initiation. Fifty hours before sacrifice, food was removed from cages. Thirty hours before sacrifice, all rats were given 250 mg fluorescein dextran by gavage (1 ml, MW 4400) ( ).

Eighteen hours before sacrifice ten rats were allowed to drink water with 5 PEG, and twelve rats were given pure water (see )

were prepared as they had been for ACF. Fluorescein-labeled cells, and nuclei (to estimate total number of cells) were then counted in 100 normal and 60 aberrant crypts at 400 magnification with a fluorescence microscope (Axiolab, Zeiss).

×

Statistical Methods

Group means were compared by Student s test, or by Welch s test when variances were not equal, or by Mann-Whitney test when data ' t ' t were not normally distributed. To compare many treated groups to a single control group, the Dunnett s test was used when Bartlett s statistics ' '

showed non-significant differences among variances. A non-parametric Kruskal-Wallis was used in other cases. Standard deviations (SD) are reported in the text, and pooled standard error of the mean (SEM) in . All values quoted correspond to two-tailed test, and value Table I P P below 0.05 was considered significant.

Results

The Effect of 19 Laxative Agents on AOM-induced ACF, and on Fecal Values

Five independent short-term studies were conducted to test the hypothesis that laxative drugs can decrease the number of ACF during the post-initiation period. The results are shown on , using the ACF values in control rats to normalize data across the five studies (full data Figure 1 not shown). PEG, unique among laxative agents, strikingly suppressed ACF. Compared to control rats, PEG-treated rats had a nine-fold reduction in the number of ACF and a 13-fold reduction in the number of large ACF (mean of 5 studies, all p<0.001). Other PEGs also reduced the number of ACF, but to a lower extent. PEG 400 was roughly half as potent as PEG 8000, as previously shown for PEG 3350 ( ). Other 6 laxatives did not reduce significantly the number of ACF ( ) except magnesium hydroxide. In bisacodyl-fed rats all crypts looked Figure 1 aberrant and colons could not be scored for ACF, but small tumors were detected (N 0.8/rat). Rats treated with the non-laxative NSAID = piroxicam had significantly less ACF than controls (p<0.001), but twice more ACF than PEG-treated rats.

Laxative polymers of high MW, including PEGs, roughly doubled both the daily fecal output (dry weight) and the fecal moisture ( , Figure 2 full data not shown). The rats consumed and excreted about 1 g of PEG per day, which explains the increased fecal weight. Small MW agents had less effect on fecal values in rats, except magnesium hydroxide (doubled fecal moisture), kaolin and paraffin oil (doubled fecal dry weight). Karaya gum, carboxymethylcellulose, polyvinylpyrrolidone and PEG were thus potent laxatives in rats (

), but only PEG 8000 strongly Figure 2 reduced the ACF number (

). This suggests that the ACF inhibition by PEG 8000 was not due solely to its fecal bulking activity. Figure 1 Indeed, a modest inverse correlation was found between the ACF number in the colon of rats and fecal dry weight and fecal moisture. The correlation coefficients were 0.40 and 0.53 respectively (p<0.05, d.f. 25). However, after exclusion of PEG data, the ACF number did not -correlate any longer with fecal dry weight or with fecal moisture.

In vitro data suggest that cytostatic effect of PEG on cell lines be due to PEG-induced osmotic pressure ( ). Osmolarity of fecal water 17 from all rats was thus measured, but it was not changed by PEG or by laxative treatments (p>0.05), and it did not correlate with ACF numbers (data not shown). These results show that PEG-prevention of carcinogenesis is not due to laxative effect, or to osmotic properties of PEG.

PEG Increased Crypt Cells Apoptosis and Proliferation

Apoptosis, an important prevention mechanism ( ), was measured by two different techniques in 3 m sections of colonic mucosa from 25 μ control and PEG-treated rats. The number of apoptotic bodies was doubled in PEG-treated rats compared with controls. After a 30-day PEG-treatment of AOM-initiated rats, the number of apoptotic bodies per crypt scored on Feulgen-fast green stained sections increased from 0.05 0.03 to 0.12 0.04 (p 0.002). In a distinct experiment, the TUNEL labeled cells per crypt increased from 0.05 0.02 in controls to 0.08

± ± = ±
0.03 in PEG-treated rats (p 0.04). In addition, PEG strikingly increased the ALP activity in feces (25-fold increase, per g of dry feces), and

± =
daily fecal excretion of ALP (44-fold increase, median of five studies). In one study, for instance, control and PEG-treated rats excreted 98 34 Crypt lengthening and cell proliferation are often associated with colon carcinogenesis ( ). We thus speculated that PEG-prevention might 27 be associated with crypt shortening and reduced cell proliferation. After a 30-day PEG-treatment in AOM-initiated rats, crypt height was reduced from 32.3 2.6 cells in controls to 27.3 2.9 in PEG-fed rats (p<0.01). But PEG surprisingly increased crypt cell proliferation. The

± ±

BrdU labeled cells per crypt increased from 3.12 0.58 in controls to 3.99 0.63 in PEG-treated rats (p<0.01). Also, the number of mitoses per ). Twice more labeled cells were Figure 3 counted per AOM-initiated aberrant crypt cells, than per normal crypt cells ( , 12.5 and 5.4 cells respectively, p<0.001). According to Table I McNeil, this might suggest that transformed cells on top of ACF are more fragile, or more exposed to mechanical stress, than normal crypt cells ( ). After fluorescent labeling, an 18-hour PEG-treatment normalized the proportion of fluorescent cells in aberrant crypts: the percentage of 21 labeled cells on ACF decreased from 12.5 to 7.0 (p<0.001), which shows that PEG eliminated cells from ACF. Indeed, PEG-treatment % slightly reduced total number of cells seen in ACF, from 42 to 38 cells per crypt (p<0.001). PEG-treatment also slightly decreased the percentage of labeled cells in normal crypts, from 5.4 to 4.0 (p<0.01).

Discussion

This study demonstrates that (i) among 19 laxatives, only PEG 8000 and its analogs were potent chemopreventive agents against colon carcinogenesis in a rat model, (ii) the mechanism of PEG protection was not related to increased fecal bulking or water content, (iii) PEG induced proliferation, epitheliosyis, and apoptosis in colonic crypts, which confirms Roy s findings ( ), and (iv) PEG seems to remove ' 28 damaged cells from the top of ACF.

The results show that a 30-day PEG-treatment reduced 9-fold the number of ACF, and 13-fold the number of large ACF (mean of 5 studies, all p<0.001). Previous studies have shown the following facts: Following a 105-day PEG-treatment rats have 20-times less ACF and 100 times less large ACF than controls ( ). PEG is more potent than most known preventive agents in this rat model ( ), and the effect is not 1 3 limited to a specific carcinogen, since hydrazine, nitrosamine, and heterocyclic amine-induced ACF are suppressed ( ). PEG effect is fast: a 6 three-day PEG-treatment, one month after carcinogen injection, halves the number of ACF in rats ( ). 6

The mechanism by which PEG can prevent carcinogenesis in rats is not known. Speculated mechanisms include (i) dilution of promoting compounds in the gut lumen due to PEG osmotic and bulking properties, (ii) protection of epithelia by PEG lubricating, coating, and sealing properties, (iii) elimination of cancer cells from the tumors by apoptosis induction. These three hypotheses are discussed here:

High-molecular weight PEGs are not absorbed from the gut of rats and bind water through hydrogen bonding ( ). In PEG-fed rats, the 29 fecal weight and moisture increased markedly ( ), and we have shown that the concentration of bile acids in fecal water is halved ( ). fig. 2 1

However, the present study clearly shows that this bulking effect does not explain the anti-cancer properties of PEG. Indeed, some laxatives increased both fecal dry weight and fecal moisture as much as PEG, but they did not reduce ACF (e.g., karaya gum). Previous studies show that psyllium and wheat bran can double the fecal bulk, but only modestly suppress ACF ( ). Magnesium hydroxide also showed some protective 3 effect here. Magnesium is a known chemopreventive agent ( ). It may act like calcium, unrelated with laxative properties ( , ). Small 30-32 33 34 tumors were detected in bisacodyl-fed rats only one month after AOM injection, thus confirming it is a strong tumor promoter ( ). To 15 conclude, because bulking does not cause chemoprevention, it should be possible to design PEG-like chemopreventive agents with no laxative effect.

Alternatively, PEG may protect the colonic mucosa by lubricating the fecal stream ( ), by providing a mucin-like barrier and reducing 29 inflammation ( ), or by facilitating the membrane resealing ( ), thus reducing the ACF over-proliferation. This hypothesis is supported by 20 21 our in vitro data ( ) and by the strong chemoprevention afforded by pluronic ( ), a PEG-like agent that binds membranes and repairs cell 17 4 membrane defects ( ). In contrast, this study clearly shows that PEG increased proliferation, like aspirin and sulindac ( , ). PEG also 22 35 36 appears to increase the turn over of cells on top of ACF crypts ( ), and greatly increased fecal ALP, a potential marker of epitheliolysis (

- table I
, ). In addition, while a 0.05 PEG concentration protects cells in vitro ( ), a 100-times higher concentration is needed to suppress ACF 23 24 % 37 in vivo ( ). 5

Lastly, according to in vitro results, PEG might eliminate transformed cells by apoptosis ( , ). This would normalize or erase ACF, 17 18 " "

explaining the fast loss of ACF ( ). Indeed, PEG-treatment doubled the number of apoptotic bodies in colonic crypts, as recently reported in 6

ACF and in Min mice ( ). This could be meaningful because a fish oil and pectin diet that increases apoptotic index by 25 decreases 28

+ %
adenocarcinoma incidence by 50 in rats ( ). In addition PEG strikingly increased epitheliolysis in the gut, increased proliferation but -% 25 reduced crypt length, and halved the number of labeled cells on top of ACF ( ). Taken together, these facts suggest that PEG has an table I abrasive effect on the mucosa. How could abrasion be protective? Although it is highly controversial, the elimination of cells from the top of crypts might be enough to stop carcinogenesis, by preventing the top-down movement of transformed cells ( ). This would explain why the The present study is not definitive, and following questions might still be addressed: (i) Do non-protective laxatives induce apoptosis and epitheliolysis? (ii) We suggest that PEG removes cells from ACF, that often hold K-mutation. The DNA of shedded cells can be detected in ras stools ( ). So, does PEG increase fecal flow of K-mutations? (iii) How could PEG induce apoptosis and epitheliolysis? Added into a 40 ras culture medium, PEG increases the osmotic pressure and shrinks cancer cells ( ). By contrast, in vivo, PEG drags water into the gut lumen, 17

which in turn balances the osmotic pressure ( ). Thus, PEG-effect is not due to osmotic pressure, but might be due to detergent properties. 29

Indeed, PEG induces cell fusion in vitro, accumulates in cancer cells, and the optimal MW to enhance endocytosis and to suppress ACF is identical, i.e., 8000 ( , ). Last, PEG-induced apoptosis may come from the modulation of transcription factors, e.g., induction of 6 41 pro-apoptotic factor Par-4, and suppression of E-cadherin down-regulating factor SNAIL ( , ). 18 42 To conclude, data suggest that PEG acts by eliminating cells from ACF. PEG is used as a mild laxative at a dose which matches the dose used in rats ( ). A recent population-based study of 1165 colonoscopies in France shows that Forlax users (a PEG 4000-based laxative) have 43 ® a halved risk of colorectal tumors ( ). High MW PEGs have no known toxicity, are not absorbed, not metabolized, and not fermented. We thus 8

suggest PEG be tested in a clinical trial.

2 %

 2 Proliferative cells in the colonic crypts were visualized immuno-histochemically using an anti-BrdU antibody ( ). Cells in mitosis and 25 apoptotic bodies per crypt were counted on 3 m sections stained by Feulgen-fast green. Apoptotic bodies were recognized based on μ morphological identification ( ). Apoptotic cells per crypt were also counted after Tdt-mediated dUTP-biotin nick end labeling of fragmented 26 DNA (Oncogene Research Products) ( ). An investigator scored 20 (proliferation) and 60 (apoptosis) fully visible crypts per rat in a blinded 25 manner.

±

  and 4158 1765 Sigma ALP Units/d respectively (p<0.001). Intestinal ALP activity in fecal water would reflect epitheliolysis ( , ). Thus, apoptosis and epitheliolysis in the mucosa.

  ± ±crypt increased from 0.35 0.19 to 0.53 0.11 (p 0.03). A duplicated study showed similar reduction of crypt length (p<0.01), and increased± ± =proliferation (p 0.04) after a 7-day PEG-treatment.=PEG Removed Cells on Top of Aberrant CryptsFluorescein dextran labeled cells were easily counted on top of normal and aberrant crypts (

Figure 1

 1 Figure 1 Among 19 laxative agents, PEG only strongly suppressed carcinogenesis in AOM-initiated rats Fischer rats (N 210) were given an injection of azoxymethane. They were then randomized to a standard AIN76 diet containing one of 19 laxative = agents (5 w/w in most cases). Aberrant crypt foci (ACF) were measured blindly after a 30-day treatment. Results from five sequential studies are % shown after normalizing values to 100 for each control groups. Area of circles matches groups size (large: 12 rats; small: 4 rats). Overlapping ' points were shifted verlapping to be more visible. Star indicates results different from control (Dunnet s test). Unnamed small points: m: mannitol, ' l: lactulose, p: psyllium, o: paraffin oil, s: sorbitol, k: kaolin. Actual values of ACF ( ) in the 5 control groups were 53 ( ), and of aberrant crypts 119 62 ( ), 96 ( ), 122 ( ), 80 ( ). Matching values in PEG-fed rats were 4 ( ), 5 ( ), 2 ( ), 22 ( ), 13 ( ). Piroxicam and bisacodyl results 139 200 264 172 8 8 6 39 23 are not shown on this figure (see text).

Figure 2 Figure 3

 23 Figure 2High MW laxative agents increased the daily fecal excretion (dry weight) and the fecal moisture (percent water) in rats fed a purified diet for one month Design: see . : different from control for fecal excretion or moisture; : different from control for fecal excretion and moisture (Dunnet s figure1* # ' test). Unnamed small points (near controls): m: mannitol, s: sorbitol, d: docusate, l: lactulose. Actual values of fecal moisture given as water ( % / ) in the 5 control groups were 30) ( ), 45 ( ), 41 ( ), 45 ( ), 44 ( ). Matching values in PEG-fed rats were and fecal dry weight, g d 0.85 0.69 0.78 1.16 1.26 68 ( ), 68 ( ), 69 ( ), 66 ( ), 68 ( ). 1.73 1.37 1.40 1.88 2.16

Table I

 I An 18-hour PEG-treatment normalizes the proportion of cells labeled by fluorescein dextran on top of aberrant crypts, 105 130 days after an injection of carcinogen AOM to rats and 30 h after a 250 mg p<0.01; Dunnet s test (normal crypts, comparison with none group) or Student s test (ACF crypts, comparison with AOM group).

			Rat Treatment	No. of rats	No. of crypts scored	Cells seen per crypt	Labeled cells per crypt	Mean percent of labeled cells
			none	6	620		14.8	0.84	5.4
	Normal Crypts
			PEG	4	460		15.2	0.67	4.0*
			AOM	6	600		14.9	1.02**	6.6
			AOM PEG +	6	680		14.8	0.66	4.1 **
	SEM						0.1	0.04	0.25
	ACF Crypts	AOM	6	350		42.4	5.64	12.5
			AOM PEG +	6	405		38.1 **	2.91 **	7.0**
	SEM						0.7	0.2	0.4
	Notes to	Table I	. SEM: pooled standard error of the mean;
	** p<0.001;
			'	"	"	'	"	"

fluorescein dextran gavage.

*

inhibition was reversible in part when treatment was discontinued ( ). Similarly, chemical peeling with PEG and salicylic acid strikingly

suppresses skin tumor development in mice ( ).

Ackowledgements:

Studies were funded by Ligue-32, INRA and DGER, Ministry of Agriculture, France.


Didier Jourdan

Nicola J Gray

Margaret M Barry

Sonja Caffe

Christophe Cornu

Fatou Diagne

El Fadi

Mychelle Hage

Sean Farmer

Michael Slade

Mychelle Y Hage

Susan M Marmot

Sawyer

Supporting every school to become a foundation for healthy lives

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The health of children and adolescents is influenced by a complex interplay of biopsychosocial, cultural, environmental, and economic factors.1 As an educational setting where children and adolescents spend a large proportion of their daily lives, school has an important influence on every student's health and wellbeing.2 Simply attending school for longer is associated with better health outcomes, including intergenerational benefits-eg, mothers who are better educated typically have healthier children than mothers who have received less education. [START_REF] Patton | Adolescence and the next generation[END_REF] We have known for more than a century that the provision of healthy food and social support at school improves attendance and increases participation in children and adolescents from disadvantaged communities.4 Globally, growing rates of school participation mean that schools are increasingly recognised as an important platform for enhancing student health, with different formal and informal mechanisms by which these health benefits can occur.5 However, there is substantial tension between the aspiration of the health sector to deliver discrete, programmatically based interventions on a range of health topics (such as nutrition, sexuality, parasite eradication, or vaccinations) with more systemic approaches that are required to influence school policies and practices, reshape environments, and build wider community partnerships that underpin sustainable health-promoting practices.6,7 If positive changes are made, schools have the potential to greatly improve the life chances of children and adolescents. Beyond academic results that influence employment and income prospects, which are themselves strong determinants of future health, schools can equip students to develop general life skills and knowledge that will benefit their health in later life. However, if positive change is not achieved then not only will students miss out on health-maximising skills and knowledge, but also their physical and mental health can be damaged by experiences at school, which can be a powerful amplifier of inequalities. Examples of institutional failings include: bullying; lack of physical activity; and poor nutrition and poor support for students with long-term health conditions (including disabilities), those from refugee backgrounds, and those who identify as LGBTI. The intense national debates about the reopening of schools during the pandemic have laid bare the interconnectedness of health and education inequalities, which can worsen when schools close.8,9 The COVID -19 pandemic is unlikely to be an isolated global health emergency; therefore, our societies and school systems must become more resilient for the future. In this Viewpoint, we will highlight the value of health professionals thinking more strategically about their roles in school health, beyond their traditional roles of providing health services and delivering curriculum material about health-based subjects. Improving the health of all students requires a focus on reducing health inequalities, which requires structural changes that health professionals can help to bring about. We will also challenge health-services researchers to consider the clash of evidence-based practice and practice-based evidence that might occur when designing and evaluating school-based studies.

Schools and health: new challenges for an old relationship

All civilisations pass down prescriptive advice about health as part of collective wisdom, and since their foundation in the 19th century, contemporary school systems have been mandated to contribute to the improvement of population health. The knowledge that positive health behaviours can be acquired in childhood and adolescence has led political authorities to assign schools the task of improving health. From lessons about hygiene, tuberculosis, and alcoholism at the end of the 19th century,10 to whole school approaches11-17 that recognise that the ethos of individual schools affects the health of its students and staff, schools have long been targets for efforts to improve health. These efforts have taken different forms at different times and must be regularly reviewed, in the context of evolving social expectations and new knowledge, to ensure that health promoting actions match the changing health needs of students. There is an emerging evidence base that shows that learning outcomes are improved, social and emotional wellbeing are increased, and health risk behaviours are reduced in response to more holistic or whole school approaches. [START_REF] Patton | Promoting social inclusion in schools: a group-randomized trial of effects on student health risk behavior and well-being[END_REF][START_REF] Langford | The World Health Organization's health promoting schools framework: a Cochrane systematic review and meta-analysis[END_REF][START_REF] Shinde | Promoting school climate and health outcomes with the SEHER multi-component secondary school intervention in Bihar, India: a cluster-randomised controlled trial[END_REF] Such approaches are typically based on coherence between the school's policies and practices that promote social inclusion and a wider commitment to education and health. The World Bank Human Capital Project recognises that the intersection of good health, nutrition, and education can drive economic growth, and that investment in children and adolescents brings substantial effects and high economic returns.21 Schools are not the only options for investment-we do not want to exclude others, including peer-led community interventions-but schools offer a basic and globally consistent setting for promoting health and wellbeing. Beyond the curriculum, providing safe spaces for girls in low-income and middle-income countries and increasing the availability of clean toilets are good examples of the type of non-programmatic responses from schools that are likely to have positive effects on student health and engagement with education, as well as school retention. Whole school approaches can be quite diverse11-17 (see examples in the panel) but share two objectives: to improve educational success, and to improve the health and wellbeing of all students and staff. The UN Sustainable Development Goals (SDGs) now form the backdrop to these initiatives.22 Among the 17 SDGs for 2030, the objectives that are most closely related to schools and health are those that link Good Health and Wellbeing (SDG3) with Quality Education (SDG4). Within the SDG agenda, explicit appreciation of the interconnected aspects of these goals also aligns with holistic approaches to student health and wellbeing. Although there is considerable evidence about the relevance of such holistic approaches (eg, WHO's Health Promoting Schools),11,19, [START_REF] Barry | A systematic review of the effectiveness of mental health promotion interventions for young people in low-and middle-income countries[END_REF][START_REF] Durlak | The impact of enhancing students' social and emotional learning: a meta-analysis of school-based universal interventions[END_REF][START_REF] Weare | Mental health promotion and problem prevention in schools: what does the evidence say?[END_REF] few countries have successfully implemented them to scale. [START_REF] Jason | Health promotion interventions[END_REF][START_REF] Who | Making every school a health promoting school[END_REF][START_REF] Herlitz | The sustainability of public health interventions in schools: a systematic review[END_REF] A crucial question is how to create the conditions for every school, in the various global educational contexts, to enact practices that positively influence health as part of the everyday life of its students and staff.29 background information that should help define the roles of health professionals and health services researchers as future partners and agents of change within schools.30 There is a large evidence base regarding the implementation of education policies and the determinants that hinder or facilitate these processes. [START_REF] Kirtman | Leadership: key competencies for whole-system change[END_REF][START_REF] Viennet | Education policy implementation: a literature review and proposed framework[END_REF][START_REF] Fuller | Global education reform: understanding the movement[END_REF] New approaches in complex education systems need to take into account the school culture, the overall coherence of the pedagogy, the constraints that affect the activity (such as the heterogeneity of students, curriculum constraints, and available resources), and the vision and usual practices of staff.34 Arguably, four interconnecting levers have to be activated to achieve educational reform: practices; structures and systems; human resources; and policies as shown in the figure.35,36 Table 1 shows how these levers might be focused for two healthrelated issues in school-namely, bullying (with its links to mental health issues) and living with diabetes. These health-related issues are globally relevant, and although there might be substantial resource differences between schools in high-income and low-income countries, they illustrate universal health and wellbeing needs of children and adolescents.

Practices

The professional practices that need to be widely adopted are those that have a positive effect on the determinants of current and future health in children and adolescents. These practices relate to three main domains: maximising educational achievement (inclusion and equity); creating an environment that is favourable to student development; and promoting general competencies and health-related skills (eg, health literacy, basic life knowledge and skills, social and emotional skills).37 These practices are linked to school management, school policy, physical and social environments, student participation and engagement, relationships with families and the community, school health and social services, and teaching.38 Curriculum and pedagogy are as important as each other in developing these practices. The example of bullying (table 1) underlines that key messages and role modelling can be conveyed to students through not only what teachers teach, but also how schools teach and engage with students more generally. Teaching that is based on specific or fragmented objectives is less able than a holistic approach to help students not only to understand the mechanisms of harassment but also to learn how to prevent or resist it.39 To maximise the motivation and agency of education professionals, and thus ensure genuine and sustainable uptake of health-promoting practices in schools, it is crucial to understand the views and practices of those professionals. Understanding the views and the practices of education professionals on health issues in schools should form the basis for the development of more realistic frameworks of practices than those that are currently in place, frameworks that are adapted to context and culture.40 These views and practices are not always shaped by initial and in-service training but are often learned in the field.41 Thus, education professionals have a range of visions of their role in health.42 Based on data from the field, there are different ways to contribute to the evolution of educational practices. The most promising ways feature collaborations between practitioners, researchers, designers, technologists, and others to develop practice-based evidence as an essential complement to the findings from other forms of education and public health research.34,43

Structures and systems The existence of supportive organisational structures at different levels, from national systems through to individual schools, is a condition for successful change. [START_REF]A guide for ensuring inclusion and equity in education[END_REF][START_REF] Weick | Educational organizations as loosely coupled systems[END_REF][START_REF] Jones | Social and emotional learning in schools: from programs to strategies and commentaries[END_REF] Much effort has been invested in education management, especially at the school level.46 A review by Samdal and Rowling suggests that effective whole school practices are supported by key implementation components, including school leadership and management practices, the school's readiness for change, and the school's organisational and support context.47 Knowing the importance of school management, individual principals are often urged to implement additional policies and programmes in various fields, even though demands on school staff are already excessive. [START_REF] Bryk | AERA distinguished lecture: accelerating how we learn to improve[END_REF] The coherence of what is required by schools, especially school management, is crucial; the notion of health as a product of quality schooling should be embedded within schools' core policies, rather than being seen as one among many other additional (ie, secondary) demands. The concept of leadership is often narrowly defined as applying solely to the principal. However, leadership is only a prerequisite for educational change at the individual school level. Schools and communities could expand leadership locally to include representatives across the key stakeholder groups. These groups include students, teachers and other school staff, parents and caregivers, as well as outside service providers. Although the involvement of health professionals will be limited by time and resource constraints,49 they can be a major asset for schools.

Human resources Evidence shows that professionalism and commitment of school staff are key elements for successful schools. [START_REF]Health education in schools: the challenge of teacher training[END_REF] The highest performing education systems tend to systematically prioritise the quality of teachers over other strategies to improve education. Current data from the Organisation for Economic Co-operation and Development for countries such as Finland suggest that high performing systems attract the most talented teachers to the most challenging classrooms, and the most capable school leaders to the most disadvantaged schools, which steers all students towards higher academic standards.51 Promoting health in all schools and reducing health inequalities is also a matter of human resources. [START_REF] Pont | Improving school leadership, vol 1: policy and practice[END_REF] Having political and institutional frameworks is necessary, but without training and capacity building for school staff, progress will stall. [START_REF] Schleicher | Preparing teachers and developing school leaders for the 21st century: lessons from around the world[END_REF] Professional development (from pre-service and in-service training) helps teachers and other school professionals acquire skills to incorporate equitable, inclusive, and health-promoting practices in their classrooms. An educator's view of their role in relation to health is a key determinant of their contribution.40 Education professionals need to be supported in the shift from a physical to a biopsychosocial understanding of health, and from a medical approach to health (seen as health professionals' core business) to a better understanding of their potential effect on health determinants.54 Studies show that teachers who have received health promotion training tend to be more frequently involved in health related projects, and have a more sustainable approach to health education, than those who have not received specific training. [START_REF] Simar | Education et santé à l'école: étude de l'impact d'un dispositif de formation et d'accompagnement sur l'implication des enseignants dans une démarche de promotion de la santé[END_REF][START_REF] Speller | Developing trainee school teachers' expertise as health promoters[END_REF][START_REF] Byrne | A longitudinal study to explore the impact of preservice teacher health training on early career teachers' roles as health promoters[END_REF] Staff training is not just an assortment of random modules; professional development in health promotion has to be part of a coherent strategy. [START_REF]Health education in schools: the challenge of teacher training[END_REF] To promote a teacher's continuing education and knowledge management, professional development must extend beyond the early stages of implementation of health programmes.57,58 Teachers who receive ongoing support through specific and timely professional development while, and after, they have opportunities to learn contextually are more likely to continue with newly introduced practices.58 Sustainable professional development is a key condition for a major shift in practices taking place within a school.59 Support from local health professionals during the design and implementation of staff development programmes can help to cement intersectoral links for mentorship and advice, and enable timely directing of students and families to local health services. Not all programmes in schools are teacher led,20 and so the human resources available to schools could be expanded to include students themselves, in peer-educator roles, or other actors within or beyond the school.

Policies In many national educational policies, health has not been a priority. Indicators of school performance rarely include health, or health education, as a key measure.60 Health improvement is one among many other competing priorities for educational systems and what schools can routinely accomplish. Time is the most scarce resource in schools, not just in low-income countries and low-resource settings, and it is not possible to add an infinite array of new programmes to what schools currently do. Given competing priorities, it is always a question of choice at the national level and at the individual school level. To ensure sustainability, health policies must be embedded within every school's core policies and be coherent with ongoing educational reform processes. In many countries, the development of school health policies can take place within two major areas of reform: the orientation toward more inclusive and equitable schools in the aim of assuring the educational success of all students; and the development of the education for sustainable development and sustainable lifestyles under the umbrella of the SDGs. The views of civil society, and other external stakeholders (especially parents and caregivers35,61), about the characteristics of a good education are very important in establishing priorities. Various groups have tried to win support for what they believe are the key problems in schools and for their proposed solutions. [START_REF]A guide for ensuring inclusion and equity in education[END_REF] The more powerful and prestigious the groups are, the more likely they are to influence school priorities. The activity of principals and teachers, and the organisation of the school systems, are directly connected to the influence of these groups.62,63 A useful example is the case of programmes that promote abstinence-only-until-marriage. These abstinence-only until-marriage programmes are scientifically and ethically problematic because a large body of evidence supports that they are not effective in delaying initiation of sexual intercourse or changing other sexual behaviours.63 Promotion of such an approach by powerful lobbies-and sometimes governments-can result in the abolition of teaching about sexuality, which then undermines comprehensive approaches to sexuality education in schools. Community expectations shape the school; depending on whether these expectations are oriented towards the wellbeing of students, academic success, or sporting success, for example, the school system will be more or less responsive to efforts to promote different aspects of physical and mental health and wellbeing. Parents' expectations are extremely influential.61 Schools' decisions are influenced by their hopes or fears about parental responses to teaching inclusivity and equitability to children, including on topics such as sexuality and gender identity.35 Communication with society is indispensable for influencing the views of education policy makers with the aim of scaling up health-promoting practices in schools. Endorsement of school policies by local health professionals can help principals and teachers to allay parental concerns, influence policy makers, and secure resources.

Experience of policy implementation in educational settings

The reviews of a large number of educational reforms or innovations, including those that were built on an evidence-based approach, are disappointing.42,64 Once implemented and evaluated, education reforms do not always produce the expected effects (in terms of student achievement, scale, or sustainability).65 As early as 1995, within the educational research literature, Tyack and Cuban30 attributed the failures of educational reforms to insufficient consideration during the implementation process of the specific school context as well as of staff practices (teachers' so-called grammar of schooling).66 Arguably, education professionals simply do not appreciate schools being seen by health policy makers primarily as a neutral platform through which to deliver interventions at scale focused on child and adolescent health. Too often, programmes focus on just a small part of school life or the curriculum and are implemented as a narrow intervention that addresses only one specific issue. Such programmes are primarily viewed by educators as having a niche or segmented role and benefit.62 Health-sector professionals who try to implement new approaches might not appreciate the overall coherence of the pedagogy, the constraints that affect the activity (such as collective management, class organisation, heterogeneity of the students, curriculum constraints, and available resources), and the vision and usual practices of staff.34 Adapting interventions to existing routines and changing contexts appears to be part of the sustainability process within schools, including for public health interventions.28 There are major differences between schools within a given education system; deviation from the norm is the general rule.67 The so-called standard school does not exist because there are differences in students' needs, rate of teacher and principal turnover, leadership, school size, location (rural or urban), and socioeconomic context. Evidence produced is always contextualised and it is often difficult to draw general conclusions that are relevant for all schools. WHO's Health Promoting School approach originated in the context of high-income countries. It was based on a liberal vision of education, a largely decentralised system that gives real autonomy to schools (eg, to implement a policy), and considers student participation, health, and wellbeing to be core objectives of the school. However, other educational cultures might value academic instruction, involvement in the community, or critical thinking more highly. Schools in low-resource settings are often extremely limited by a scarcity of skilled workforce and funding, and in many centralised contexts the autonomy of schools is minimal. A context-sensitive approach is essential for improving the capacity of schools to promote child and adolescent health, and is a prerequisite for health professionals and researchers who wish to make advances therein. The challenge is to create the best conditions for sustainable changes across different geographical and cultural settings.

Producing and sharing knowledge Studies produced in the context of controlled interventions are especially valuable in showing proof of principle, in comparing outcomes from different models, and in understanding cost-effectiveness. However, the necessary evidence to support the scaling up and sustainability of holistic approaches to health in schools is not limited to data produced in the experimental contexts that are well known to, and arguably most valued by, health professionals and health-services researchers. Programmes that work in one setting often do not work in another.42 Programmes supported through external interventions or dedicated grants might not last in their entirety (or at all) once the funding ends. [START_REF] Herlitz | The sustainability of public health interventions in schools: a systematic review[END_REF] The double translation process from international guidelines such as the WHO Health Promoting School approach, to national and local policies has been shown to be highly complex.68 There is a need for inclusion of a wide range of relevant and robust evidence in this global discussion,69 such as evidence about: practices to promote health in school, contexts and cultures, determinants of inequalities, change within the educational systems, and factors affecting the implementation of health-promoting practices.36 Different kinds of data are required to examine each area.70 A reasoned understanding of which data can best be produced in experimental conditions, and which data can best suit observation in everyday practice, is required. In all cases, combining scientific quality and relevance for professionals will be needed. Valuing what is already done, and sharing best practices and improving them using the evidence produced by research, are relevant approaches (eg, the recommendations from the Aspen Institute National Commission on Social, Emotional, and Academic Development,71 and Sherer and colleagues72). Without such a strategy, there is a high risk of having a minimal effect on the educational service offered to students. More multidisciplinary research that combines the strengths and traditions of health and education with meaningful codesign with teachers and students will also benefit the field.

Building bridges between education and health: the role of health professionals Schools need a truly intersectoral partnership

to maximise their potential in shaping children's and adolescents' health knowledge, attitudes, behaviours, and health outcomes. The levers to optimise the health of children and adolescents are shared by families, communities, policy makers, and professionals from health, social, and education sectors. The involvement of health professionals can be a powerful asset in gaining support for whole school endeavours. Since the population often views health from a medical or disease orientation, support from health professionals and health policy makers is, arguably, one of the main enablers of the development of the contribution of schools to health. The question is how health professionals can support schools at national and local levels to move beyond the rhetoric and make real, sustainable changes to strengthen every school's capacity as a healthy place to live, learn, and work.2 The current COVID-19 pandemic has brought unprecedented attention to schools, education, and health. Attention has extended well beyond infection control against severe acute respiratory syndrome coronavirus 2, to wider appreciation of the effect of school closures on student and parent mental health and wellbeing. This attention provides new opportunities to develop closer working relationships between health and education professionals in schools, and effective local partnerships between health and education professionals have been reported to have contributed to the successful reopening of schools.73 During the pandemic, it is pleasing to see the breadth of roles that have been articulated for health professionals, such as school nurses, who can "assess the data available, integrate it into prior knowledge about infection control, capitalise on emergency preparedness planning, advocate for equitable distribution of services, access evidence-based resources, plan for interventions in the schools, and constantly evaluate outcomes to improve approaches".74 Table 2 shows multiple ways in which health professionals can make a concrete contribution to intersectoral actions. Of course, the commitment of health professionals depends on the context, including the nature of the activity, the existence of an appropriate participation framework, and the availability of practical support required to free up time to perform these roles. One simple example of a strategy that health professionals can use to make an intersectoral contribution is by asking children and adolescents (and parents or caregivers) during health consultations, whether within a doctor's office or a community pharmacy, about what they have learnt at school regarding a particular health topic, and how it was different or similar to what they had learnt at home. Health professionals, whether in a doctor's office or a community pharmacy, can also show that they value what health promotion is being done in schools, which means knowing what is being done in schools. The contribution of health professionals must move from a narrow focus on children with specific health issues (eg, HIV/AIDS, or asthma), to all students. Likewise, sharing information with students and school principals about the range of disease prevention and health promotion approaches that are being undertaken within different sectors, including schools and health services, can achieve greater intersectoral understanding within the systems that support health and learning.

Health professionals as catalysts for healthy changes in schools Supporting every school to become a foundation for healthy lives means creating the conditions for schools to be inclusive (every child in school), equitable (ensuring the success of all children), and healthy (providing a learning environment that promotes health both now and in the future). The core business of schools is focused on increasing educational outcomes rather than the reduction of health problems, despite knowledge of how closely these objectives are intertwined.4 Schools should not be considered simply as convenient platforms for the implementation of prevention programmes, or as spaces to deliver expert knowledge about the importance of lifestyle for preventing diseases. Data show that when schools are considered only as convenient platforms or spaces to introduce interventions, there is a high risk that the intervention might increase inequalities. [START_REF] Qadri | School-based oral health education increases caries inequalities[END_REF] The challenge is for better appreciation of the multiplicity of ways that health promotion can be understood as a means for schools to achieve educational goals. To have a substantial effect on health promotion in the school setting, stronger alliances between education and health professionals must be created at various organisational levels. At a global level, initiatives seek to learn lessons from the enactment of successful reforms and change in education, and contribute to forging a common culture to support schools, including in their efforts to promote student health. These goals are shared by global initiatives such as the UN interagency collaboration for a new approach to school health known as "Stepping up effective school health and nutrition",7 the Global Standards for Health Promoting Schools (a joint initiative between UNESCO and WHO),76 and the UNESCO Chair and WHO Collaborating Centre in Global Health and Education.77 Wider collaboration between international and national professional groups and coalitions that are mindful of the challenges noted in the literature and described here offers a genuine roadmap to production of action-oriented knowledge and to sharing of improvement strategies. Notwithstanding these efforts to promote health, greater cooperation and mutual support of local health and education professionals is essential to achieve our global ambition for schools to be the means through which the world's children and adolescents develop the capabilities for lifelong health, wellbeing, and success. Health professionals' support for school activities related to health is a key factor for sustainable health-promoting practices in schools. Rooted in their communities, cognisant of the prevailing culture and context, and intimately involved in the lives of local families, health professionals represent a formidable force as we ride this next wave of change.

Search strategy and selection criteria

We searched PubMed for relevant papers from Jan 1, 1980, to Aug 31, 2020, using the search term "health in schools". Papers and books were also identified through searches of our own files and publications. Only publications in English and French were included.

The final reference list was based on the relevance to the broad scope of this Viewpoint.

Contributors

  DJ and NJG conceptualised the article and led the writing of the paper with SMS. All authors contributed to drafting and revising the manuscript, and approved the final draft. Declaration of interests SMS reports grants from WHO on Health Promoting Schools during the writing of the Viewpoint, and was also a member of WHO's School Health Services Guideline Development Group. The other authors declare no competing interests. Acknowledgments MYF is associated with NCD Child. NCD Child is supported through grants to the Hospital for Sick Kids (SickKids, Toronto, Canada), from the AstraZeneca Young Health Program and Novo Nordisk. SickKids also provides in-kind contributions to support the NCD Child Secretariat. DJ and NJG are associated with the UNESCO Chair Global Health and Education and WHO Collaborating Centre for Research in Education and Health. UNESCO Chair Global Health and Education and WHO Collaborating Centre for Research in Education and Health are supported by the non-profit organisations MGEN (Mutuelle Générale de l'Education Nationale, France) and Groupe VYV (France). Université Clermont-Auvergne (Chamalières, France) provides in-kind contributions to support the UNESCO Chair secretariat.

  Jourdan et al -Text only version of the Lancet Child and Adolescent Health article -Embargoed until 3 February, 2021

Jourdan et al -Text only version of the Lancet Child and Adolescent Health article -Embargoed until 3 February, 2021


O Krebs

A Lemaître

Optically induced coupling of two magnetic dopant spins by a photoexcited hole in a Mn-doped InAs/GaAs quantum dot

Keywords: numbers: 78.67.Hc, 75.50.Pp, 71.70.Gm, 75.10.Dg

We report evidence of a photo-induced coupling between two spins provided by Mn dopants in their neutral acceptor state A 0 in a single InAs/GaAs quantum dot. The coupling occurs due to simultaneous exchange interactions between each of the two dopant spins and a photo-created hole. Micro-photoluminescence spectroscopy achieved both in longitudinal and perpendicular magnetic field reveals the splitting of the 4 spin configurations |J1 = ±1, J2 = ±1 due to the 2A 0 -hole exchange interaction. We obtain a comprehensive interpretation of the experimental data with a simplified spin Hamiltonian model, which more specifically shows that the hole-mediated coupling is similar to a ε12 -70 µeV exchange interaction between both A 0 spins.

Semiconductor quantum dots (QDs) have been extensively studied in the last decade to address and manipulate optically the spin of a single trapped electron or hole [1][2][3][4][5][6][7]. The controllable coupling of two such spins, aimed to perform quantum operations, is more challenging as it requires to deal with the indiscernibility of identical carriers. A solution consists in isolating both carriers in two distinct QDs which can be tunnel-coupled by a tunable electric field [8][9][10][11]. Alternatively, an individual QD containing two point-like spins, each provided by a single atom and with negligible mutual interaction, could be used to control their coupling through their respective interaction with a common electron or hole. This could be achieved with magnetic dopants indirectly coupled via the sp-d exchange interaction. Such magnetically doped QDs would be of particular interest because the strength of the exchange, as reported for singly Mndoped CdTe/ZnTe [12] or InAs/GaAs [13] QDs, is high enough to spectrally resolve the different magnetic spin states, and therefore to achieve their optical manipulation and readout, notably with few-picosecond pulses.

The optical spectroscopy of CdTe/ZnTe QDs doped with two Mn atoms has been recently reported [14]. It revealed clear spin correlations induced by their overlapping with a common resident or photo-created carrier in the quantum dot and the possibility for optical control. Here, we present an analogous investigation of an InAs/GaAs QD containing two Mn atoms, relying on the magneto-optical spectroscopy in a longitudinal or perpendicular magnetic field and its interpretation within a simplified spin Hamiltonian model. Our results show that the magnetic dopants experience an effective exchange interaction induced by a photo-created hole. Besides many conceptual similarities with the II-VI system, the specific properties of Mn-doped III-V QDs [15,16] make the interpretation somewhat simpler with essentially 4 spin configurations (against 36), more suitable for the design of a spin-based CNOT gate.

In the III-V In x Ga 1-x As compound the magnetic dopant provided by a substitutional Mn atom forms an acceptor state denoted A 0 consisting of a negative center A -on the Mn site and a bound hole in the host valence band with ∼1 nm Bohr radius [17]. The direct exchange interaction between two such acceptors must typically extend over twice their radius, in agreement with the direct imaging of 2A 0 wave-functions in GaAs subsurface [18]. In an InAs/GaAs QD of about 20 nm diameter and 4 nm height, the direct coupling between two randomly distributed A 0 should vanish in more than 95% of the cases. The effective spin of a magnetic acceptor A 0 is associated to its total angular momentum J = 1 corresponding to the anti-ferromagnetic configuration of the Mn 3d 5 electrons (spin S = 5/2) and the hole angular momentum J h = 3/2 [19][20][21]. In a self-assembled InAs/GaAs QD presenting both lens shape and biaxial strain, the state |J z = 0 of the J = 1 triplet, where z is a Mn-site-dependent direction close to the growth axis z, is substantially shifted at higher energy (counting positively the hole energy) with respect to the states |J z = ±1 by a few meV [15,22]. Consequently, this level is negligibly occupied at low temperature ( 2 K) and one generally observes the A 0 acceptor mostly in one of the two states |J z = ±1 . We used the same Mn-doped photo-diode device as in Ref.'s 13 and 16 which consists of a QD layer embedded in between an electron reservoir and a semi-transparent Schottky gate enabling us to control the charge. The setup for micro-photoluminescence (µ-PL) spectroscopy in magnetic field is described in the same references. PL spectra presented in this Letter were acquired at low temperature (T ≈ 1.7 K) under non-resonant excitation (HeNe 633 nm laser line) in a magnetic field applied in the Faraday or Voigt configuration, i.e. parallel or perpendicular to the z optical axis. In both cases, the PL signal was measured in σ + circular polarization corresponding to the detection of a | ⇑ -spin hole recombining with a | ↓ -spin electron. The sample was studied in a regime where the QDs are charged by an electron, so to the σ + signal in Faraday configuration. The magnetic fields at which the anticrossings δ take place (positive in A 0 -X - state, near zero in A 0 -e state) determine the position of the characteristic X-pattern.

that negative trions X -(2 electrons, 1 hole) are photocreated under illumination. Since both electrons in X - are paired in a singlet, their spin dependent properties vanish, which allows us to regard a negative trion as a single hole.

During an experimental run performed to measure a 1Mn-doped quantum dot (QD1 in figure 1) we observed another set of spatially and spectrally correlated lines (QD2), which could be ascribed from its magnetic dependence to a 2Mn-doped QD. First we use QD1 to recall the typical features of a 1Mn-doped QD revealed by a magnetic field in Faraday geometry [13,15,23]. In Fig. 1(b), the PL spectra consist of two main lines (labeled |± 1 ) which are split by an energy ∆ and experience each two anticrossings δ with two weaker lines. Altogether they form a characteristic X-pattern. The second noteworthy feature is the PL intensity transfer from | + 1 to | -1 (|-1 to |+1 ) in a strong positive (negative) field. Both these observations can be understood with the help of Fig. 1(d) which illustrates the field dependence of the | ± 1, ⇑ or ↑ energy levels in the initial A 0 -X -and fi-nal A 0 -electron (or A 0 -e) states. The X-pattern results from the δ anticrossing of the | ± 1 A 0 states due to their anisotropic coupling [13,22]. Due to the different strengths of the A 0 -e and A 0 -h exchange interactions, the δ anti-crossing between the |± 1 states occur at different magnetic fields for the A 0 -X -and A 0 -e states. For A 0 -e, it remains near zero field because of small exchange interaction (usually below δ), while for A 0 -X -state, it is shifted to the positive magnetic field B ≈ ∆/(2g A 0 µ B ), where g A 0 denotes the effective g-factor of A 0 spin J = 1 and µ B is the Bohr magneton [13,15]. Near these anticrossings, optical transitions with A 0 spin-flip are partially allowed (dashed lines in Fig. 1(d)) which produces the X-pattern. At higher magnetic fields, A 0 magnetization, i.e. the thermal relaxation between |± 1, ⇑ or ↑ Zeeman-split levels (wavy arrows in Fig. 1(d)), results in the pronounced change in intensity of the corresponding |± 1 lines in Fig. 1(b).

The magnetic dispersion of QD2 PL spectrum (Fig. 2(a)) reveals 4 bright lines exhibiting features quite similar to those of 1Mn-doped QDs (X-pattern and magnetization in high fields). However, the splittings denoted ∆ 1 and ∆ 2 appear twice, and so do the X-patterns X i (i = 1, 2) which connect each ∆ i -split pair of lines with faint lines anticrossing the bright lines at

B i = ∆ i /(2g A 0 i µ B )
. This observation strongly differs from the X-pattern duplicate already observed in 1Mn-doped QDs due either to electrostatic fluctuations of the environment or to different charge state [13]. The fact that there is two different X-patterns and that the magnetization takes place within the 4 bright lines as a whole (i.e. only the highest (lowest) line survives in high positive (negative) field) strongly supports the interpretation that QD2 is doped with two distinct acceptors A 0 i (i = 1, 2), each characterized by an exchange splitting ∆ i and an anticrossing energy δ i . Within this framework, the 4 bright lines can be associated to the 4 acceptor state configurations |J z 1 ,A 0 1 = ±1, J z 2 ,A 0 2 = ±1 as indicated in Fig. 2(a). The spin Hamiltonian model discussed below fully agrees with this interpretation.

To determine the proper local strain tensor experienced by each acceptor, we measured the effect of the transverse magnetic field B x . As previously reported for 1Mn-doped QDs [15], the Zeeman-split lines exhibit a strong circular polarization up to high magnetic fields both for QD1 (Fig. 1(c)) and QD2 (Fig. 3(a)). This is evidenced by the asymmetry of the spectra with respect to B x . This property results from the exchange interaction between A 0 and the QD hole which acts as a giant effective magnetic field in the z direction (close to z) associated with the strain experienced by A 0 , and thus inhibits the hole spin mixing by the transverse field [15]. Conversely, the electron Zeeman effect is much less perturbed by the small A 0 -e exchange, which yields (↑ ± ↓)/ √ ing the ∆ 2 -split lines (thus analogous of X 2 in Faraday) which develop in negative fields. Much fainter lines crossing each other in the -4 T field region (where they almost vanish) likely form two other X-patterns connecting the ∆ 1 -split lines (analogous of X 1 ). For QD1 (Fig. 1(d)), we observe in contrast a single X-pattern which moreover develops in positive fields. Since in Voigt geometry the X-pattern position in the magnetic field is determined by the local strain tensor experienced by A 0 [15], one can conclude that both Mn-doped QDs do not share a common A 0 [START_REF]A common A 0 located between two close-lying QDs could possibly interact with the evanescent part of the hole wavefunctions in both QDs[END_REF], even though they appear in the same µ-PL spectra. Simulation of QD2 spectra can thus be achieved by assuming that QD2 contains two distinct A 0 in its own volume.

In previous works, we developed a general spin Hamiltonian model in which A 0 dopants were described as two interacting spins provided by the 3d 5 electrons (S = 5/2) and the Mn-bound hole (J h = 3/2) [13,15]. Here, for simplicity, we treat each A 0 acceptor as an effective spin J = 1. It reduces the Hilbert space dimension of the A 0 pair from 576 (24×24) to 9 (3×3), and turns out to be quite sufficient in moderate magnetic field to reproduce the acceptor levels in the observed J = 1 configuration. Our model still includes a strain-induced zero field splitting of the J = 1 states specific to each A 0 i (i = 1, 2), which formally reads [15,[START_REF] Yakunin | [END_REF]

Ĥstr = 2 i=1 -2∆ 0 i 3 Ĵ2 z i - 1 2 Ĵ2 x i + Ĵ2 y i + δ i 2 Ĵ2 x i -Ĵ2 y i .
(1) The first term in Eq. 1 amounts to a tensile strain along the z i direction (defined by arbitrary polar angles θ s,i and azimuthal angles φ s,i ) blue-shifting by ∆ 0 i the |0 level of A 0 i from the |± 1 ones. The latter are further split by δ i due to the second term of Eq. 1 which amounts to a shear strain in the plane perpendicular to z i . For the QD-confined hole, a similar Hamiltonian (but without inplane shear strain) is used to reproduce the few 10 meV ∆ HL splitting between the heavy-hole (J h,z = ±3/2) and light-hole (J h,z = ±1/2) states [15]. The Zeeman effect is included for all individual spins : the hole and electron g-factors are deduced from the linear dispersion of the bright lines in Voigt and Faraday measurements, to g h = 0.65 and g e = -0.4, respectively, while both A 0 g-factors are fitted to reproduce the slopes of the faint lines in the X-patterns. Heisenberg-type exchange interactions, ε h,i Ĵi • Ĵh or ε e,i Ĵi • Ŝe (i = 1, 2), are assumed between both A 0 i effective spins and the hole or electron spin, respectively. A specific anisotropic exchange Ĥa,i =

ρ i ε h,i ( Ĵ2 h,z i Ĵi • Ĵh + Ĵi • Ĵh Ĵ2 h,z i
) is also taken into account to include the hole valence band mixing at the A 0 i sites [26]. This term which introduces the anisotropy strength ρ i and the direction z i from the QD center to the A 0 i site (defined by angles θ a,i and φ a,i ), is required only to reproduce the dark-bright splitting ∆ DB observed around ±3.5 T (Fig. 2(a)) [13,15]. The total spin Hamiltonians of the initial (2A 0 -X -≡ 2A 0 -hole) and final (2A 0 -electron) states are each diagonalized for a series of magnetic fields as reported in Fig. 2(b)-(c) and Fig. 3(b)-(c). Note a specific color scale, which highlights the levels contributing to σ + polarized recombination, is used to encode the hole spin J h,z and electron spin S e,z . From the arrays of calculated eigenvalues and eigenvectors, we can generate the theoretical PL emission spectra simulating the experimental ones, by following the procedure described in Ref. 15 in order to include A 0 thermalization, diamagnetic shift and experimental broadening. The fitting parameters used in Fig. 2(d) and Fig. 3(d) are presented in Tab. I. Despite the simplicity of the acceptor description as a J = 1 spin, this model reproduces accurately the experimental observations in both field configurations with a single set of parameters. Note however the A 0 g-factors have to be taken explicitly anisotropic (g A 0 ,⊥ = g A 0 ,|| ), whilst in a more elaborated model the sole strain-induced fine structure (Eq. 1) was enough to explain the magnetic anisotropy of 1Mn-doped QDs [15]. Since the A 0 i -hole exchange is large compared to the A 0 i -electron exchange (i.e. 3|ε h,i | |ε e,i |) [13,16], as confirmed by the weakly perturbed electron Zeeman splitting in Voigt configuration, we can unequivocally determine the exchange parameters from the size and position in magnetic field of the X-patterns. Note that without the anisotropic term Ĥa,i , we expect that In Voigt geometry (Fig. 3(b),(c)), the magnetic dispersion is reduced due to the QD anisotropy, but is still dominated by the 2A 0 Zeeman effect associated with the finite projection of the external field B x onto the actual quantization axis z i of A 0 i spin [15]. The hole keeps a pronounced J h,z = ±3/2 character up to high fields thanks to the large 2A 0 -induced spin splittings ≈ |∆ 1 ± ∆ 2 | of a few 100 µeV's. Conversely, for the electron, the small 2A 0 -electron exchange (ε e,i ∼ 20 µeV) compares with the transverse Zeeman term only up to about 2 T. In this low field region, the spectral details (see e.g. the marked lines in Fig. 3(a),(d)) provide sensitive criteria to finely adjust the corresponding ε e,i parameters, in agreement with the estimate from the Faraday measurements.

|∆ i | ≈ |3ε h,i -ε e,i |
A remarkable feature of such a 2Mn-doped quantum dot is to provide a hole-mediated coupling between both A 0 spins. Indeed, at zero magnetic field (see Fig. 2(b) or 3(b)), the exchange interaction with the photo-created hole results in a ferromagnetic ground state | ± 1, ±1 . Thermalization of the 2A 0 spins in the presence of the hole induces thus a positive spin-spin correlation χ 12 = Ĵ1 • Ĵ2 , as would be produced by a ferromagnetic exchange between the A 0 spins. By assuming a Boltzmann distribution, we calculated χ 12 as a function of temperature for both the hole-mediated coupling, and a direct exchange ε 12 Ĵ1 • Ĵ2 between A 0 spins (with ε h,1 = ε h,2 = 0) [14]. A good agreement between the two correlation functions was obtained in the 0-10 K temperature range with ε 12 -70 µeV. For comparison, the actual direct exchange |ε 12 | is estimated in QD2 to be less than 5 µeV [28]. Note in our measurements there is no obvious spin-spin correlation in the zero-field PL spectrum, likely because of the X -lifetime and the A 0 effective temperature of about 10 K. A more favorable regime to observe this correlation should be reached at a gate voltage where the quantum dot is charged by a resident hole.

In conclusion, we demonstrate the experimental coupling of two acceptor-type magnetic dopants by a photocreated hole in an InAs/GaAs quantum dot. Our study relies on the quantitative analysis of the magneto-PL images from a quantum dot doped with 2 Mn atoms by using a simplified spin Hamiltonian model. It shows that both neutral magnetic dopants behave each like a 2-level systems giving rise altogether to 2×2 spin configurations. Combining this feature with the possibility to optically control a single spin [16] would open a way to achieve an optically-triggered spin-based CNOT gate. Besides, the hole-mediated coupling between the magnetic dopants also opens an original approach to optically investigate Zener-type ferromagnetism at the nanometer scale.

FIG. 1 .

 1 FIG. 1. (a) µ-PL spectrum in zero magnetic field showing two sets of correlated lines (dashed line frames) originating from a 1Mn-doped (QD1) and a 2Mn-doped (QD2) quantum dot. (b),(c) Density plots of QD1 PL intensity measured in σ + polarization as a function of the magnetic field in Faraday and Voigt configuration respectively (E0 = 1.2702 eV). (d) Sketch of the most significant energy levels which contribute for QD1to the σ + signal in Faraday configuration. The magnetic fields at which the anticrossings δ take place (positive in A 0 -X - state, near zero in A 0 -e state) determine the position of the characteristic X-pattern.

  2 spin states split by ∼ |g e µ B B x |, see e.g. Fig. 1(c). For QD2 (Fig. 3(a)), we again observe two X-patterns connect-

FIG. 2 .

 2 FIG. 2. (a) Density plot of QD2 PL intensity measured in σ + polarization as a function of the magnetic field Bz in Faraday configuration (E0 = 1.274 eV). Dashed lines are drawn to make visible the X-patterns (X1) associated to the anticrossings at B1. (b),(c) Energy levels of 2A 0 -X -and 2A 0 -e calculated as a function of Bz and drawn with a color scale proportional to J h,z and Se,z, respectively. (d) Theoretical density plot of the corresponding X -PL intensity in σ + polarization, assuming T A 0 = 11 K.

FIG. 3 .

 3 FIG. 3. (a) Density plot of QD2 PL intensity measured in σ + polarization as a function of the magnetic field Bx in Voigt configuration (E0 = 1.274 eV). (b),(c) Energy levels of 2A 0 -hole and 2A 0 -electron calculated as a function of Bx and drawn with a color scale proportional to J h,z and Se,z, respectively. (d) Theoretical density plot of the corresponding X -PL intensity in σ + polarization, assuming T A 0 = 8 K.

  which is verified for A 0 2 since ρ 2 = 0[27]. With the help of Fig.2(b),(c), one can now identify the four 2A 0 spin configurations giving rise to the four bright lines in the σ + magneto-PL image : two ferromagnetic levels |±1, ±1 which rapidly shift with the field, and two anti-ferromagnetic | ± 1, ∓1 levels with a weaker magnetic dispersion (due to the sole hole or electron Zeeman term). The origin of the four X-patterns becomes clear in Fig.2(b): each anticrossing δ i experienced by the acceptor A 0 i appears twice in the level diagram due to both possible orientations | ± 1 of the second dopant A 0 j =i .

TABLE I .

 I Parameters used for theoretical simulations. ∆ 0 i in meV, δi, ε h,i and εe,i in µeV, θs,i, φs,i , θa,i, and φa,i in degrees ( • ).

	∆ 0 i δi θs,i φs,i g A 0 i ,|| g A 0 i ,⊥ ε h,i εe,i ρi θa,i φa,i
	A 0 i=1 5 40 30 130	4	2.2 -380 25 -0.24 95 -100
	A 0 i=2 3.5 35 25 -120 4	3.2 -55 15	0	-	-

anisotropic term only.[28] When included in the model, the direct exchange |ε12| induces a relative magnetic shift of the two similar Xpatterns (X1 or X2) by ε12/(g A 0 ,|| µB), which is not observed experimentally.