CONTRIBUTION OF MULTISPECTRAL AUTOFLUORESCENCE IMAGING TO HISTOCHEMISTRY IN UNDERSTANDING SORGHUM INTERNODE HYDROLYSIS PATTERN

* cecile.barron@inrae.fr Sorghum is the world's fifth largest cereal crop and exhibits high biomass yield potentials. This cereal is suitable to support the development of biomass value chains in temperate and semi-arid environments. Digestibility of plant feedstock is one of the key properties and is largely dependent on the overall lignification of the plant which is monitored at the tissue scale by the relative amounts and composition of plant organ. To obtain details on organ anatomy and spatial distribution of main components, medium-throughput histochemical methods have been developed on maize and sorghum internodes [1,2]. FASGA staining procedure is based onthe competition between 2 dyes and reveals regions with different staining colour (essentially red and blue color) which may be interpreted in terms of tissue ligninification [3]. In addition to staining procedures, the specific spectral properties of phenolic compounds could also be exploited to map their distributions using spectral imaging technics. Multispectral autofluorescence imaging was successfully applied to maize stem internode to reveal differences in cell wall phenolic compounds distribution (lignin and hydroxycinnamic acids) [4]. The objective of this work was to evaluate the relevance of multispectral autofluorescence imaging to better understand contrasted hydrolysis patterns observed inside the sorghum stem internode.

Two sorghum genotypes differing in their hydrolysis yield were selected. Internodes (at the bottom of the stem) from four healthy plants harvested at the grain filling stage were characterized for histochemistry in relation to spatial-susceptibility to enzymatic hydrolysis. Cell wall digestion was realized with a combined cellulase/hemicellulase 4% mix during 72h. The hydrolyzed sections were stained with FASGA and the hydrolysis yields were estimated by comparing the area of the section before and after hydrolysis (Figure 1A,B).. Images were analyzed with the open-source ImageJ freeware4 and a dedicated script to quantify anatomical traits. Multispectral autofluorescence images (Figure 1C) were acquired using a multi-zoom macroscope AZ100M (Nikon, Japan) equipped with 4 filter blocks to acquire images under UV, blue and green excitation lights. Emission light was recovered through long pass filters and, taking advantage of the RGB channels of the color camera, each image was split in three channels and stack alltogether to obtain a 12 channels multispectral image. In such multispectral images, each pixel is characterized by an intensity profile that can be considered as a spectral signature (Figure 1D).

Observed after FASGA staining, both genotypes showed typical sorghum internode organization with two main anatomical zones: (1) a mainly red-stained peripheral zone Z1 (rind), which is rich in vascular bundles and sclerenchyma cells and is surrounded by an epidermis on the most external part, and (2) an inner zone Z2 (pith), composed of vascular bundles spread into blue-stained parenchyma. As already observed, no hydrolysis of the redstained zone (Z1) was evidenced for these two genotypes. Different hydrolysis pattern in the low-lignified zone (Z2) was revealed. One genotype showed a limited losse of material (below 10% for two plants and up to 25 % for the other two plants). For the second genotype hydrolysis yields were about 40-50%, corresponding to an almost complete degradataion of the Z2 region. For both genotypes, the hydrolysis was more pronounced in the middle of the internode cross section.

Comparison of the brightfield images with the color representation of the average multispectral images shows that all the cell wall showed autofluorescence under UV or visible excitation. Differences in color in this multispectral image highlight the main tissues with highly lignified tissue appearing as pink/orange. No visual difference between parenchyma was evidenced and more precise analysis of spectral signature was carried out. Spectral signature of blue-stained parenchyma cell walls were extracted in both hydrolysed and not hydrolysed regions. Whole spectral signatures were compared through principal component analysis. Slight differences were observed, in particular under UV-excitation light and must be faced to overall spectral variability (within a cross section and between plants).

The relevance of combined classical histochemical and multispectral imaging was emphasized in this study allowing to highlight the different behaviours of constratsing genotypes and different regions of the internodes.

Figure 1 :

 1 Figure 1 : Cross section of sorghum maize stem internode. A,B. Fasga staining before (A) and after (B) digestion. C. Color representation of the average multispectral autofluorescence image. D. Autofluorescence pseudospectrum from parenchyma cell walls.

Acknowledgments

This work was supported by Biomass For the Future project (ANR-11-BTBR-0006-BFF) funded by the French National Research Agency (ANR).


Pretrel Hugues

Vaux Samuel

Experimental and numerical analysis of fire scenarios involving two mechanically ventilated compartments connected together with a horizontal vent

This work deals with an experimental and numerical investigation of fire scenario involving two rooms mechanically ventilated and connected together with a horizontal vent. The objective is to investigate the effect of a horizontal opening on a fire scenario and especially on the burning rate. The study is based on the analysis of a set of large scale fire experiments performed in the framework of the OECD PRISME-2 project in the DIVA multi-room facility of the Institut de Radioprotection et de Sûreté Nucléaire (IRSN), and of numerical simulations performed with the ISIS CFD code developed by IRSN. The fire scenario consists of two rooms, one above the other, mechanically ventilated, and connected to each other with a horizontal vent of 1 m 2 . The fire is heptane pool fire located in the lower room. The analysis focuses on the coupling between the burning rate, the flow at the vent and the configuration of the mechanical ventilation. Several regimes of combustion are encountered from well ventilated steady fire to under-ventilated unsteady and oscillatory fire. The results show that the burning rate is controlled by both the mechanical

ventilation and the downward flow from the vent. The numerical simulations highlight the specific pattern of the oxygen concentration field induced by the downward flow at the vent.

KEYWORDS

INTRODUCTION

The propagation of smoke and hot combustion products is an issue of practical importance for fire safety assessment. In many industrial situations, smoke, responsible for the transport of heat and soot particles, may lead to the failure of safety systems. In the particular domain of nuclear industry, typical events related to smoke that should be prevented are the clogging of highefficiency (HEPA) filters located in a ventilation network or the malfunction of electrical or electronic devices. The understanding and the development of appropriate modeling for smoke flows are of practical importance in the field of fire safety assessment.

The understanding and the prediction of fire scenario involving smoke flows through horizontal openings remain a challenging exercise (Figure 1). Indeed, as for doorways, the flow at a horizontal opening section may be bi-directional, induced by buoyancy and the mass conservation condition. However, because of the horizontal section, normal to the direction of gravity, the vent flow shows a much more complex behavior than the one for a vertical section, as doorway or window. The flow at the vent may experience several regimes, depending on the pressure and temperature differences conditions at the orifice: one-directional upward, bi-directional upward and downward, and one-directional downward. In addition, the bi-directional flow regime shows a complex flow pattern characterized by a time variation of the upward or downward flows locations in the vent section. One challenging issue is the fire scenario involving a fire compartment equipped with a vent for which a strong interaction may occur between the fire and the vent flow. In case of a bi-directional flow regime at the vent, the downward contribution may have a substantial influence on the combustion process, by modifying the flow rate of oxygen entering the fire enclosure, Figure 1. Brown et al. [START_REF] Brown | Natural convection through rectangular openings in partitions[END_REF], Epstein et al. [START_REF] Epstein | Combined natural convection and forced flow through small openings in a horizontal partition, with special reference to flows in multicompartment enclosures[END_REF], [START_REF] Epstein | Buoyancy-driven exchange flow through small openings in horizontal partitions[END_REF], Heskestad et al. [START_REF] Heskestad | Inflow of air required at wall and ceiling apertures to prevent escape of fire smoke[END_REF] and Tan et al. [START_REF] Tan | Flow through horizontal vents as related to compartment fire environments[END_REF]. Based on small-scale experimental approaches, these studies led to a theoretical description of the flow for a pure buoyancy bi-directional flow condition, based on the Froude number, and of the purge conditions, when mechanical ventilation is applied. From these works, Cooper [START_REF] Cooper | Calculation of the flow through a horizontal ceiling/floor vent[END_REF]- [START_REF] Cooper | Calculating combined buoyancy-and pressure-driven flow through a shallow, horizontal, circular vent: Application to a problem of steady burning in a ceiling-vented enclosure[END_REF] developed empirical correlations, predicting the gas flow rate for different flow regimes, from pure natural convection to mixed convection, with the final aim of introducing them in zone fire models. Recently, additional works were both performed for the validation of the Cooper's models (Pretrel et al. [START_REF] Pretrel | Smoke flow through a horizontal vent separating two mechanically ventilated rooms from large scale fire tests[END_REF]) and the vent flow characterization, using an advanced measurement technique, the Stereo PIV (Varrall et al. [12] [13]).

The interaction between the vent flow and the smoke propagation in the compartments have been studied, pointing out the consequences of the upward flow on the smoke stratification in the upper room, as well as in the lower room (Hagglund et al. [START_REF] Hagglund | An experimental study of the smoke spread in a two plane compartment[END_REF] Pretrel et al. [START_REF] Pretrel | Smoke induced flow in two rooms mechanically ventilated and linked with a horizontal vent type opening[END_REF]- [START_REF] Prétrel | Experimental study based on largescale smoke propagation fire tests through a horizontal opening connecting two mechanically ventilated compartments[END_REF], Dai et al. [START_REF] Dai | Buoyancy-Driven Flow in a Two-Story Compartment[END_REF], Li [START_REF] Li | Characteristics of Buoyancy Driven Natural Ventilation through Horizontal Openings[END_REF]). Recent works have emphasized the consequences of the vent flow on the fire and especially on the fire burning rate, for the natural ventilation scenario, with the fire room only connected to the outside with a horizontal vent (He et al. [START_REF] He | Ventilation limited extinction of fires in ceiling vented compartments[END_REF], [START_REF] He | Vent size effect on self-extinction of pool fire in a ceiling vented compartment[END_REF], [START_REF] He | Pool Fires in a Corner Ceiling Vented Cabin: Ghosting Flame and Corresponding Fire Parameters[END_REF], Chen [START_REF] Chen | Investigation of the burning mechanism in an enclosure fire with a horizontal ceiling vent[END_REF]). For these typical configurations, additional experimental and theoretical researches are needed to improve the general understanding of these flows and their consequences of a fire.

The present contribution focuses on the behavior of a compartment fire scenario, involving a vent and the consequences on the fire, for the specific configuration of closed and mechanically ventilated enclosures. This work extends the He's studies, conducted in natural ventilation, and considering forced ventilation. The present objectives are to improve the knowledge of fire scenario involving rooms, linked with vent and in particular to investigate the effect of three specific scenario of ventilation on the burning rate and the vent flow. It carries on previous studies performed with constant and controlled burning rate with gas propane fire (Audouin et al., [START_REF] Audouin | Experimental and Numerical Study of Smoke Propagation Through a Vent Separating Two Mechanically Ventilated Rooms[END_REF] Pretrel et al. [START_REF] Pretrel | Smoke flow through a horizontal vent separating two mechanically ventilated rooms from large scale fire tests[END_REF], [START_REF] Pretrel | Smoke induced flow in two rooms mechanically ventilated and linked with a horizontal vent type opening[END_REF], [START_REF] Prétrel | Experimental study based on largescale smoke propagation fire tests through a horizontal opening connecting two mechanically ventilated compartments[END_REF]). The present study is based on fire tests involving heptane pool fire whom the burning rate varies in time depending on the vent flow.

This study is based on the analysis of large scale fire tests performed in the framework of the Vertical Smoke Propagation, VSP, campaign of the OECD PRISME2 project and is supported by numerical simulations performed with the ISIS CFD code, developed by IRSN. First the fire tests are described. Then, the analysis focuses on the typical behavior of the fire scenario, the regimes of burning rates and the flow at the vent. Numerical simulations are then presented in order to better describe the influence of the downward flow on the oxygen concentration field in the fire room.

DESCRIPTION OF THE TEST CAMPAIGN

Experimental facility

The facility was the DIVA facility of IRSN, identical to the one of previous works with propane gas fire [START_REF] Prétrel | Experimental study based on largescale smoke propagation fire tests through a horizontal opening connecting two mechanically ventilated compartments[END_REF]. As illustrated in Figure 2 The fire source was a 0.4 m 2 heptane fuel pool fire located in the lower room. The pool was a circular, 13 cm in height metallic pan, filled with a given amount of fuel before the test. Ignition was performed with a propane gas burner. The amount of fuel diminished during the combustion phase. Two positions were considered, either at the centre of the room, either at the North-West corner, as indicated in Figure 2 and Figure 3.

Measurements

Heptane mass was measured during the experiment with a SARTORIUS weighting balance accurate within 2 g. The fuel mass loss rate was then determined from the time derivative of the mass. The ventilation flow rates were measured with average Pitot probe devices, located in the ventilation ducts and connected to membrane pressure transducers.

The vent was fitted with 13 bi-directional velocity probes, together with K-type thermocouples in a cross-section of the vent as detailed in [START_REF] Prétrel | Experimental study based on largescale smoke propagation fire tests through a horizontal opening connecting two mechanically ventilated compartments[END_REF]. The two rooms were equipped with four thermocouple trees, named SW, CC, NE and SE for the upper room L4, and SW, CC, NW and NE for the fire room L3. Each mast were equipped with nine K-type thermocouples located at 0.05 m, 0.55 m, 1.05 m, 1.55 m, 2.05 m, 2.55 m, 3.05 m, 3.55 m and 3.90 m from the ground. The oxygen, carbon dioxide and carbon monoxide concentrations were measured inside the room at three locations (upper and lower part on the SW tree and near the pool) and in the admission and exhaust branches. The gas analyzers EMERSON XSTREAM and SIEMENS ULTRAMAT apparatuses measured concentration of dry gas sample. A time delay of about 20 seconds was determined and then corrected for the analysis and post processing of the data.

The measurement uncertainties are shown in Table 1. They were evaluated from calibration tests, carried out before or after the tests and using the data provided by the manufacturers of the sensors. The calibration tests consisted in obtaining the differences between standard values (from specific calibrated sensors or samples) and the measured values, read on the acquisition system. Two levels of uncertainties were defined. The standard uncertainties, obtained at room temperature, without fire. A second level, the expanded uncertainty, took into account the fire test conditions by introducing a coverage (or correction) factor, applied to the standard uncertainty. This coverage factor balance fire effects that can significantly influence the measurement results. The flow regime at the vent may vary from pure natural convection (bidirectional flow) to forced convection (on-directional). The pressure difference, from both sides of the opening and prior to ignition, is the parameter that characterized the flow regime. It is computed as with Av=1.061 m 2 the total vent area and CD=0.74, the vent flow coefficient determined experimentally. The variable is the volumetric flow rate at the vent determined from the mass balance before ignition, as detailed in [START_REF] Pretrel | Smoke flow through a horizontal vent separating two mechanically ventilated rooms from large scale fire tests[END_REF] and [START_REF] Prétrel | Experimental study based on largescale smoke propagation fire tests through a horizontal opening connecting two mechanically ventilated compartments[END_REF]. The flow rate and the pressure difference prior to ignition are indicated in Table 2. The experimental procedure was as follows. First the ventilation network was powered up to achieve the targeted ventilation flow rates. Then the heptane pool was ignited. A mixed convection flow started to occur at the vent, and smoke progressively filled the adjacent room. The fire extinguished by lack of fuel (PR2_VSP_2 test) or lack of oxygen in the room (the other tests).

EXAMPLE OF A TYPICAL BEHAVIOUR

The typical behavior of such real fire scenario is presented from the PR2_VSP_2 test with the ventilation configuration A3/E4. The burning rate of the 0.4 m 2 heptane pool shows a nearly steady behavior at about 23 g/s (cf. Figure 4). Before ignition, the air flow rates at the admission of room L3 and at the exhaust of room L4 are equal at about 0.75 kg/s (cf. Figure 5). Then, flame propagation leads to an expansion of the gas, an increase of the exhaust flow rate and a decrease of the admission flow rate. Once the steady behavior is recovered, the two flow rates become equal again. After 10 minutes of fire, the fire starts becoming unstable leading consequently to fluctuating behaviors of the flow in the ventilation branches (see Figure 5). These fluctuations correspond to unstable behavior of the burning rate during this period (cf. Figure 4). The admission flow rate is not sufficient to compensate the oxygen consumption by the fire and the oxygen concentrations in the two rooms decrease continuously until a steady situation is reached (Figure 6). The oxygen concentrations in the upper room gives the lowest value (about 10%-vol), and is very homogenous in the room with no significant difference between the upper and the lower part (O2_L4_BAS and O2_L4_HAUT in Figure 6). The concentration in the fire room give higher levels (about 12%-vol) because of the fresh air brought by the admission line. The oxygen concentration measured at the vent is surprisingly higher than those in the two rooms. It is explained by the short distance between the vent and the inlet section that may induce local effect of fresh air entrance.

The smoke propagation within the two rooms leads to a specific vertical temperature distribution, illustrated in Figure 7. Along the fire plume axis (masts CC_L3 and CC_L4 in Figure 7) the temperature decreases from the flame temperature level (up to 800°C) toward about 450°C at the vent section and then 250°C at the ceiling of room L4. Away from this axis, the temperature profiles in the two rooms show an opposite variation with an increase from the ground to the ceiling (masts SW_L3 and SW_L4 in Figure 7). These profiles illustrate typical thermal stratification with higher temperature at the top of each room. For this test, the flow at the vent is mainly one directional upward, with nearly no downstream flow coming from the upper room as indicated in Figure 8. This behavior is mainly due to the ventilation configuration (admission in the lower room and exhaust in the upper room) that tends to avoid any entrance of fresh air from the upper room.

The fire heat release rate is also computing, directly from the fuel mass loss rate (HRR_M) and with chemicals methods: HRR_OC for the oxygen consumption method and HRR_CDG for the carbon dioxide generation method, [START_REF] Prétrel | Experimental study based on largescale smoke propagation fire tests through a horizontal opening connecting two mechanically ventilated compartments[END_REF]. For this test, the average is about 900 kW and the three methods give similar results.

The analysis of the other tests shows similar trends. The main differences concern the burning rate and its interaction with the flow at the vent.

SEVERAL BURNING RATE REGIMES

The time variation of the burning rate (also named fuel mass loss rate) is presented for the four tests in Figure 10. Three particular behaviors are observed and discussed: the reduction of burning rate with the ventilation flow rate, the oscillatory behavior and the identification of critical pool area.

Reduction of the burning rate with the ventilation

During the first instants (about 200 s) after ignition, the burning rate shows very similar behavior whatever the tests; the external environment around the fire place being similar. Then after this period, the external environment is modified because of the change of the rate of ventilation in the fire room and also the ventilation configuration. As a result, different behaviors of the burning rate are observed.

For the highest ventilation rate of the lower room (PR2_VSP_2 test, TrL3=20.4 h -1 , i.e. =2356 m 3 /h), the burning rate gives the highest level (about 23 g/s). This value is close to the one expected in open atmosphere (about 22 g/s, i.e. 54 g/s/m 2 obtained with the Babrauskas relationship, [START_REF] Babrauskas | Estimating large pool fire burning rates[END_REF], with =100 g/s and =1.1 m -1 ). Then, extinction occurs after total combustion of the mass of fuel.

With a lower ventilation flow rate (PR2_VSP_4BIS test, TrL3=13.4 h -1 , i.e. =1546 m 3 /h), the burning rate decreases to about 18 g/s, but only during a short period and then increases again rapidly up to 40 g/s followed by self-extinction due to lack of oxygen (fuel remains in the pan).

For the two lowest rates of ventilation, (PR2_VSP_3 and PR2_VSP_4 tests, TrL3=8.5 h -1 , i.e. =988 m 3 /h), the burning rate shows either a rapid self-extinction (PR2_VSP_3 test) or an oscillatory behavior followed by extinction due to lack of oxygen. The reduction of the burning rate with the ventilation flow rate is typical to compartment fire scenario. This phenomenon depends, among other parameters, on the reference global equivalent ratio GERo defined as the ratio between the rate of oxygen that is required for the combustion in open atmosphere, , and the rate of oxygen brought to the enclosure by the mechanical ventilation, , before ignition. The normalized fuel mass loss rate, , diminishes with the increase of the GERo (Figure 11) as expected and demonstrated theoretically for well-stirred reactor [START_REF] Pretrel | Well-stirred reactor approach for interpreting the combined effect of oxygen and temperature on the steady burning rate of pool fire in a confined and ventilated enclosure[END_REF]. For the present data, different regimes of burning rate are identified depending on the range of GERo. For GERo lower than 0.5, the burning rate is similar to the one in open atmosphere (M*~1). In the range GERo = [0.6 ; 1.3], the dimensionless burning rate decreases and the combustion regime can be said "under-ventilated". Above GERo = 1.3, there is extinction, and the burning rate cannot be maintained. These limits are orders of magnitude determined from the present data. Between these two limits, there is a range [1 ; 1.3] where the burning rate is very unstable and oscillatory behaviors are observed. These regimes are very similar to those identified in natural ventilation by He & al [START_REF] He | Ventilation limited extinction of fires in ceiling vented compartments[END_REF]. In the present study with mechanical ventilation, the governing parameter GERo includes the ventilation flow rate, whereas in the He's study; the governing parameter is the ventilation parameter, , considering the vent size Av only.

In the present study, the two parameters affecting the burning rate are the renewal rate of the fire room, TrL3, (and thus GERo) that characterized the air brought by the ventilation but also the pressure difference at the vent, Po. This former variable specifies the likelihood of seeing downward flow at the vent and therefore the level of additional oxygen brought by the vent to the fire room. This additional effect explains the differences between the two tests PR2_VSP_3 and PR2_VSP_4 that show different results but with the same GERo. These tests have the same

TrL3, but two different values for Po.

Oscillatory behavior

For the PR2_VSP_3 test whom the conditions are close to the extinction limit, three large oscillations of the burning rate are observed with an average frequency of 6.7 10 -3 Hz (average period of 148 s) and large amplitude of about 20 g/s for an average value of 14.8 g/s. These are typical to low frequency behavior and very similar to fire oscillations encountered in one forced ventilated room without vent [START_REF] Pretrel | Experimental and numerical study of low frequency oscillatory behaviour of a large-scale hydrocarbon pool fire in a mechanically ventilated compartment[END_REF]. They are explained as a coupling between the burning rate, the pressure in the room that governs the oxygen entrance and the convective time for the oxygen to reach the burning area. This oscillatory behavior can also be related to several recent studies showing the occurrence of oscillatory fire and ghosting flame phenomena with vent in naturally ventilated room, without mechanical ventilation [START_REF] He | Ventilation limited extinction of fires in ceiling vented compartments[END_REF], [START_REF] Chen | Analysis of compartment fires with a ceiling vent[END_REF]. The present tests demonstrate that similar unsteady behaviors can be encountered between scenarios with or without mechanical ventilation or/and vent. The common features are the under-ventilated conditions, specified among others parameters with GERo, but also the capability of having a strong coupling between the burning rate and an admission of air (naturally or forced through a vent or a regular admission pipe) associated with a time delay.

Critical pool size leading to self-extinction

When dealing with compartment fire equipped with ceiling vent, an important issue for the design is the critical vent area under which no steady combustion is maintained and self-extinction occurs, because the flow rate of oxygen coming from the vent is not sufficient to ensure combustion. In natural convection, without mechanical ventilation, recent studies with pool fires show that the critical vent size is about 2 to 10 times higher than the pool area [20] [27]. Applying this criteria to the present study, a critical vent size of 1 m 2 corresponds to a range of pool fire area of [0.1 ; 0.5] m 2 . The 0.4 m 2 pool fire considered in the present tests corresponds therefore to a critical pool size that could have led to self-extinction without mechanical ventilation (natural ventilation case). The present data confirms that self-extinction can be expected if the ventilation flow rate is low (test PR2_VSP_4). If the ventilation flow rate is increased as it is done for the other tests, steady combustion phase can be achieved due to the additional feed of oxygen.

CONSCEQUENCES ON THE VENT FLOW Flow regimes

The flow at the vent is governed by temperature and pressure differences from both side of the opening. With no pressure difference, the flow is bi-directional with equal flow rate in both directions. With a non-zero pressure difference, induced by the forced ventilation for instance, the flow is unbalanced. For pressure difference above the critical pressure (or purge pressure), the flow becomes one -directional upward or downward according to the sign of the pressure difference. For the present tests with variable fire heat release rate, the vent flow varies with time following the time variation of the fire. The upward and downward mass flow rates are presented in Figure 12. The four tests with three ventilation configurations, A3/E4, A34/E34 and A34/E3, are sorted according to the initial pressure difference at the vent, Po.

For test PR2_VSP_2 with A3/E4 configuration, the flow is mainly upward and nearly no downward flows are measured (Figure 12). The corresponding initial vent pressure difference is 0.41 Pa. The test PR2_VSP_3 with Po=0 Pa leads to bidirectional flow with equal upward and downward flows similar to a situation encountered for natural ventilation. This result confirms that the reduction of the initial pressure difference contributes to decrease the upward flow and to increase the downward flow. This result demonstrates also that bidirectional regime at the vent, corresponding to natural convection case, can be encountered with mechanical ventilation scenario, provided the ventilation is set-up with no pressure difference at the vent, which is the case with the ventilation configuration A34/E34. The effect of the pressure difference at the vent is also noticed on the velocity field. Figure 13 compares the velocity profiles in one diagonal of the vent. With no initial pressure difference at the vent, Po = 0 Pa for test PR2_VSP_3, the velocity profile shows a form with equal downward and upward flows. The increase of the initial pressure difference contributes to unbalance the flow with an increase of the upward flow and a reduction of the downward one. The initial pressure difference at the vent is an appropriate parameter to characterize the flow regime and the distribution between the upward and downward flow.

Contribution of the downward flow to the fire room

An important issue for the understanding of the burning rate variation is to assess the contribution of the downward flow to the total amount of air available for combustion in the fire room. From Figure 15, it is noticed that the test PR2_VSP_4 with configuration A34/E4 leads to the lowest total amount of air entering the fire rooms. This result is in accordance with the time variation of the burning rate that shows rapid extinction due to lack of oxygen.

This results show that the reduction of the pressure difference at the vent prior to ignition favors the increase of the downward flow at the vent that can be a catalyst for unstable oscillatory behaviors of the burning rate.

DOWNWARD FLOW ANALYSIS FROM NUMERICAL SIMULATIONS

Numerical simulations of the fire tests have been performed in order to improve the physical description of the vent flow and especially the downward flow feeding the fire room in oxygen.

Numerical software ISIS

The ISIS1 software, computational tool based on low-Mach number approximation, was especially developed for simulations of fires in mechanically ventilated compartments. The Reynoldsstress tensor and turbulent scalar fluxes are modelled using the eddy viscosity hypothesis and the first-order k -e model with two balance equations. Standard wall functions are used to take into account the boundary layers as the two-equation closure model is no more valid near the walls, where viscous effects are predominant. Turbulent combustion is based on the infinitely fast chemistry conserved scalar approach, using the mixture fraction and the fuel mass fraction.

The mean reaction rate, controlled by the turbulent flow mixing, is determined by the Eddy-Dissipation Combustion (EDC) model [START_REF] He | Ventilation limited extinction of fires in ceiling vented compartments[END_REF]. A one-step irreversible combustion reaction for the fuel is considered and involves oxygen and products in the presence of a neutral gas. The model for the soot production and transport is based a simple approach considering a constant soot conversion factor, νs in the single one-step reaction [START_REF] He | Vent size effect on self-extinction of pool fire in a ceiling vented compartment[END_REF]. The radiative transfers are dealt with the finite volume method [START_REF] He | Pool Fires in a Corner Ceiling Vented Cabin: Ghosting Flame and Corresponding Fire Parameters[END_REF] assuming a gray and non-diffusive media. The gas absorption coefficient of the mixture used the total emissivity approach of the weighted sum of gray gases model (WSGGM) and the soot absorption coefficient is related to the soot volume fraction according to the Mie theory. The wall conduction is taken into account through the 1D Fourier's equation and the convective flux is given by standard laws based on laminar and turbulent

Prandtl numbers [START_REF] Chen | Investigation of the burning mechanism in an enclosure fire with a horizontal ceiling vent[END_REF].

Concerning the boundary conditions, the experimental mass loss rate is imposed for the fire source area, and classical wall boundary conditions are applied on the walls.

Validation of the ISIS simulation

The numerical simulations are compared to experimental data in order to assess the capability of prediction of the flows in both room and at the vent. The variables to be compared are gas temperature in the fire room, the volume flow rates in the ventilation and the mass flow rate at the vent.

First the prediction of the gas temperature in the fire room is compared to the experimental data in Figure 16. The simulations reproduce satisfactorily the amplitude of the temperature as well as the vertical thermal stratification in the fire room with a nearly constant vertical gradient of temperature. This result demonstrates the capability of the ISIS code to reproduce the temperature field in the fire room which governs then the buoyancy flux at the vent.

Then, the comparison between the simulations and the experiments for the flow rates in the ventilation is presented in Figure 17

Flow pattern induced by the downward flow

In order to better describe the way the downward flow is transported in the fire room, numerical simulations of the flow field in the fire room are investigated. The analysis of the oxygen concentration fields superimposed with the velocity field is presented in Figure 18 for two tests PR2_VSP_3 and PR2_VSP_4 for which the downward flow is significant. The downward flow is characterized by higher oxygen concentrations and by vertical downward velocities that can reached at least the mid-height of the fire room and nearly the ground level for test PR2_VSP_3

(Figure 18 -a). These velocity fields attest that the downward flow is not re-entrained by the smoke in the vicinity of the vent but can deeply penetrate the lower room and contributes to feed the burning regions with "fresh" air. This result may be an element that explains the oscillatory behavior of the burning rate. The way the oxygen is brought to the fire room by the downward flow, possibly with a time delay, may be a condition for unsteady behavior of the burning rate. This study has highlighted that the fire scenario involving mechanical ventilation and a vent remains a challenging issue both experimentally and numerically.

Figure 1 :

 1 Figure 1: Illustration of a typical fire scenario involving a horizontal opening linking two compartments. Pioneering works on vent flow have been conducted by Mercer et al. [1] [2], Perry et al. [3],

  , it consisted of two superposed rooms connected together by a horizontal opening or vent. The room dimensions were as follows: 4 m in height × 5 m × 6 m = 120 m 3 for the lower room, named "L3", and 4 m in height × 5 m × 8.5 m = 170 m 3 for the upper room, named "L4". The two rooms were linked by a rectangular vent of 1,030 mm × 1,030 mm = 1,061 m 2 , centered in the fire room. They were mechanically ventilated by an industrial network, with admission and exhaust branches located in the upper part of each room. The ventilation inlets and outlets within the room were positioned so that they did not influence the flow at the horizontal opening; the directions are indicated by red and blue arrows in Figure 3.

Figure 2 :

 2 Figure 2: Pictures and drawings of the facility

Figure 3 :

 3 Figure 3: Schematic illustration of the three ventilation configurations Test matrix and experimental procedure Four fire tests, named PR2_VSP_2, PR2_VSP_3, PR2_VSP_4 and PR2_VSP_4BIS were performed and the test parameters are shown in Table 2. The test parameters were the position of the fire source in the fire room (either centre in the room or off-centre in the North West corner), the ventilation configurations, named A3/E4, A34/E34 and A34/E3, and the ventilation flow rate. The ventilation configuration specified different manner of blowing air within the two rooms, as illustrated in Figure 3. The configuration A3/E4 consisted of one admission (inlet) in lower room L3 (named "A3") and one exhaust (outlet) in upper room L4 (named "E4"). The configuration A34/E34 consisted of one admission and one exhaust in both rooms L3 and L4. The configuration A34/E4 consisted of one admission in both rooms L3 and L4 and only one exhaust, in upper room L4. The ventilation flow rate was expressed with a renewal rate parameter, Tr. Two definitions have been considered. The global renewal rate, Tr, defined as the ratio between the sum of the admission's volumetric flow rate (in both rooms), before fire ignition and the total volume of the two rooms, )/V where (V = 120 + 170 = 290 m 3 ) and the renewal rate of the fire room, TrL3, based on the room L3 parameters : /VL3.

  The four tests mimic different situations of flow at the vent. The first test, PR2_VSP_2, favored a situation with dominant upward flow at the vent because of no exhaust line in the fire room and fire position below the vent. The pressure difference at the vent is maximum Po=0.41 Pa. The second test, PR2_VSP_3, favored a situation with significant downward flow at the vent because of equilibrium of pressure and a limitation of local momentum imposed by the fire with its position away from the vent. The pressure difference at the vent prior to ignition is nul Po=0 Pa and corresponds to a regime of natural convection. The two tests, PR2_VSP_4 and PR2_VSP_4BIS, correspond to intermediate situations between the two first configurations. The test, PR2_VSP_4BIS, investigated the effect of the ventilation flow rate, in comparison to the PR2_VSP_4 test, by increasing the total renewal rate. The four tests proposed, therefore, several combinations for the two ventilation parameters (Po, TrL3), with increasing pressure difference

Figure 4 :Figure 5 :

 45 Figure 4: Time variation of the fuel mass loss rate for the test PR2_VSP_2

Figure 6 :Figure 7 :

 67 Figure 6: Time variation of the oxygen concentration at three positions in the two rooms L3 and L4 (low (BAS), near the pool (FOYER) and upper (HAUT)) and at the vent (L3L4) Figure 7: Vertical temperature profiles at the axis of the vent (CC) and on the SW axis in the fire room L3 (z=[0;4]m) and in the upper room L4 (z=[4;8]m) at t=500s.

Figure 8 :Figure 9 :

 89 Figure 8: Time variation of the upward (UP) and downward (DO) air mass flow rate at the vent

Figure 10 :

 10 Figure 10: Time variation of the fuel mass loss rate for the four tests

Figure 11 :

 11 Figure 11: Normalized mean fuel mass loss rate versus the reference global equivalent ratio GERo

Figure 12 :Figure 13 :

 1213 Figure 12: Time variation of upward and downward mass flow rate at the vent for the four tests Figure 13: Vertical velocity profiles in a diagonal of the vent at t=200s for the four tests

Figure 14

 14 Figure 14 presents the time variations of the downward flow rate at the vent, of the admission flow rate, from the ventilation and of the sum of the two terms. The tests are characterized by

Figure 15 presents

 15 Figure 15 presents the average contribution of each term over the whole duration of the combustion phase versus the initial pressure reference at the vent. The ventilation configuration A3/E4 (test PR2_VSP_2 with [Po, TrL3]=[0.41 ; 20.4]) is the one that brings the largest amount of air, mostly by the ventilation, with nearly no air coming from the vent. This configuration leads to the larger level of burning rate with steady regime. The configuration A34/E34 (test PR2_VSP_3 with [Po, TrL3]=[ 0.0 ; 8.5]) is the one that gives equal contributions from the admission line of the ventilation and from the vent, but also the largest amount of downward flow. This configuration corresponds to the test where oscillatory burning rate are encountered. The downward flow is then an important criterion in the occurrence of oscillatory of the burning rate.

Figure 14 :

 14 Figure 14: Time variation of the mass flow rates entering the fire room by the ventilation (MADM_L3) and by the vent (MDO_VENT) for the four tests

Figure 15 :

 15 Figure 15: Mass flow rate entering the fire room by the ventilation (M adm) and by the vent (Mdown vent) versus the initial pressure difference at the vent Po

  (left columns). The simulations reproduce satisfactorily the experiments with similar trends. The increase of the exhaust flow rates and the decrease of the admission flow due to the temperature rise in the enclosure are correctly captured by the simulations. This result indicates that the temperature fields within the room are globally well predicted by simulations.The comparisons of mass flow rates at the vent are also presented the three tests Figure17(left columns). There is also a good agreement between experiments and simulations. The three regimes of flow at the vent are correctly predicted by the simulations: mainly one directional upstream for test PR2_VSP_2, bidirectional with equal contribution in both directions for test PR2_VSP_3 and an intermediate regime for test PR3_VSP_4 (bidirectional but with a dominant upward flow in comparison to the downward flow). This result demonstrates that the equilibri-14 um between the buoyancy that governs the bidirectional regime and the forced convection that tends to unbalance this regime is well captured by the simulations. With the fuel mass flow rate (and thus the fire heat release rate) as an input condition, the simulations predict satisfactorily the mixed convection flow fields induced by buoyancy and inertia. This validation on the flow field gives credit on the simulations in order to analysis the behavior of the downward flow within the fire room.

Figure 16 :

 16 Figure 16: Comparison between the experimental and numerical gas temperature in the fire room at three times for the test PR2_VSP_3.

Figure 17 :

 17 Figure 17: Comparison between the experimental and numerical flow rates in the admission and exhaust branches of the ventilation (left columns) and at the vent (right columns) for the three tests PR2_VSP_2 (one admission in room L3 and one exhaust in room L4), PR2_VSP_3 (one admission and one exhaust in both rooms) and PR2_VSP_4 (two admissions in rooms L3 and L4 and one exhaust in room L4)

Figure 18 :

 18 Figure 18: Superimposed gas velocity field and oxygen concentration field : (a) and (b) PR2_VSP_3 test, AA and BB plans at two times, (c) and (d) PR2_VSP_4 test, AA and BB plans at two times,

  : fire, horizontal opening, vent, under-ventilation, compartment

	NOMENCLATURE LISTING
	A	Vent Area (m 2 )	Greek	27
	CD	Discharge coefficient (-)		Density (kg/m 3 )
	GER	Global equivalent ratio (-)	subscripts
		Mass flow rate (kg/s)	adm, A	Admission
	M	Dimensionless mass flow rate (-)	ext, E	Exhaust
	r	Stoichiometric ratio (-)	up	Upward flow
	P	Pressure difference (Pa)	do	Downward flow
		Volume flow rate (m 3 /h)	L3	Fire room L3
		Heat release (W)	L4	Adjacent upper room L3
	Tr	Renewal rate (h -1 /h)	*	Dimensionless
	Y	Mass fraction	∞	Ambient atmosphere
			f	Fuel or fire
			air	Air
			v	Vent
			O2	oxygen
			o	Initial or reference

Table 1 -

 1 Uncertainty assessment of physical quantities

	Physical variable	Range	Standard uncertainty	Coverage	Expanded uncertainty
				factor
	Mass	[0-300] kg	0.002 kg	2	0.004 kg
	O2 molar fraction	[0; 0.25] mol/mol	0.0025mol/mol	1	0.0025mol/mol
	CO2 molar fraction	[0; 0.10] mol/mol	0.001mol/mol	1	0.001mol/mol
	CO molar fraction	[0; 1000] ppm	5 ppm	2	10 ppm
	Pressure transducers	[-2500; +2500] Pa	4 Pa	2	8 Pa
	K type thermocouple	[0;1300] °K	2°K	1	2°K
	Air flow rate	[-2500;+2500] m3/h	100 m 3 /h	2	200 m 3 /h

Table 2 -

 2 Test parameters and ventilation conditions measured before ignition

	Name		PR2_VSP_2	PR2_VSP_3	PR2_VSP_4	PR2_VSP_4BIS
	Fire position		C	Off-C	Off-C	Off-C
	Ventilation configuration		A3/E4	A34/E34	A34/E4	A34/E4
		m 3 /h	2356	988	948	1546
		m 3 /h	0	1048	0	0
	Tr L3	h -1	20,4	8,5	8,2	13,4
		m 3 /h	0	1451	1366	1324
		m 3 /h	2371	1460	2373	2967
	Tr L3+L4	h -1	8,1	8,4	8,0	9,9
		m 3 /h	2338	-25	987	1611
	Po	Pa	0,41	0,00	0,07	0,20

C=centre in the room; Off-C= off-center in North-West corner

The CFD code ISIS can be freely downloaded from https://gforge.irsn.fr/gf/project/isis/.


PharmD Alexandre Pachot
email: alexandre.pachot@eu.biomerieux.com

PharmD Guillaume Monneret

PhD Aurélie Brion

PharmD Fabienne Venet

MD Julien Bohé

PhD Jacques Bienvenu

PhD; Bruno Mougin

PhD Alain Lepape

H Thizy

Messenger RNA expression of major histocompatibility complex class II genes in whole blood from septic shock patients*

D

espite Ͼ20 yrs of extensive research, severe sepsis and septic shock remain the leading causes of mortality in critically ill patients [START_REF] Angus | Epidemiology of severe sepsis in the United States: Analysis of incidence, outcome, and associated costs of care[END_REF]. Over the past 2 decades, the results of clinical trials using antiinflammatory therapies have been disappointing [START_REF] Vincent | Clinical trials of immunomodulatory therapies in severe sepsis and septic shock[END_REF][START_REF] Abraham | Why immunomodulatory therapies have not worked in sepsis[END_REF]. Although the host response to an infection consists in a quasisimultaneous release in the blood of proand antiinflammatory mediators, the systemic antiinflammatory response rapidly predominates in many patients [START_REF] Munford | Normal responses to injury prevent systemic inflammation and can be immunosuppressive[END_REF]. This sustained immunosuppressive profile, called "immunoparalysis," is primarily characterized by T-cell anergy and a defect in antigen presentation [START_REF] Oberholzer | Sepsis syndromes: Understanding the role of innate and acquired immunity[END_REF][START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF]. Although the direct link between immunoparalysis and sepsis-associated morbidity and mortality has not been clearly demonstrated thus far, it is well established that critically ill patients with persistent immunoparalysis show an increased risk of contracting a nosocomial infection [START_REF] Asadullah | Very low monocytic HLA-DR expression indicates high risk of infection-Immunomonitoring for patients after neurosurgery and patients during high dose steroid therapy[END_REF][START_REF] Muehlstedt | Increased IL-10 production and HLA-DR suppression in the lungs of injured patients precede the development of nosocomial pneumonia[END_REF], which might dramatically worsen their prognosis. The failure of antiinflammatory treatments appears to be the result of, at least in part, the lack of tools allowing a precise characterization and staging of patients [START_REF] Abraham | Why immunomodulatory therapies have not worked in sepsis[END_REF].

The decreased expression of human leukocyte antigen-DR (HLA-DR) on monocytes constitutes a reliable marker of immunoparalysis [START_REF] Manjuck | Decreased response to recall antigens is associated with depressed costimulatory receptor expression in septic critically ill patients[END_REF][START_REF] Wolk | Impaired antigen presentation by human monocytes during endotoxin tolerance[END_REF] and seems to correlate with an increased risk of a fatal outcome [START_REF] Muller Kobold | Leukocyte activation in sepsis: Correlations with disease state and mortality[END_REF][START_REF] Docke | Monocyte deactivation in septic patients: Restoration by IFN-gamma treatment[END_REF][START_REF] Monneret | Analytical requirements for measuring monocytic human lymphocyte antigen DR by flow cytometry: Application to the monitoring of patients with septic shock[END_REF]. Immune monitoring by studying monocyte HLA-DR expression might therefore help to guide medical decision making [START_REF] Docke | Monocyte deactivation in septic patients: Restoration by IFN-gamma treatment[END_REF][START_REF] Kox | Immunomodulatory therapies in sepsis[END_REF]. Hence, understanding the molecular basis of immunoparalysis represents an important challenge to establish causal therapies and prevention strategies for this clinical situation. The decreased expression of HLA-DR in monocytes observed in the course of septic shock remains poorly understood. The reendocytosis and intracellular sequestration of major histocompatibility complex class (MHC) II molecules was recently described in purified monocytes [START_REF] Fumeaux | Role of interleukin-10 in the intracellular sequestration of human leukocyte antigen-DR in monocytes during septic shock[END_REF]. This phenomenon appears to be mainly mediated by interleukin 10, which plays a major role in sep-Objective: The decreased expression of human leukocyte antigen (HLA)-DR on monocytes is proposed as a major feature of sepsis-induced immunodepression. The objective of the present study was to investigate, in whole blood from septic shock patients, the messenger RNA (mRNA) expression of a gene panel, which is essential to ensure major histocompatibility complex class II protein structure, transport, and peptide loading.

Design: The authors conducted a cohort study.

Setting: This study was conducted in intensive care units at a university hospital.

Subjects: The study included septic shock patients (n ؍ 41) and healthy volunteers (n ؍ 15).

Measurements and Main Results: Using quantitative reverse transcriptase-polymerase chain reaction, we found that the highly polymorphic HLA-DRB1 and nonpolymorphic-DRA mRNA levels were significantly decreased in whole blood from patients with septic shock compared with healthy volunteer both on days 1-3 and 4 -10 after the onset of shock. This profile was also observed for genes encoding the invariant chain, the [START_REF] Fumeaux | Role of interleukin-10 in the intracellular sequestration of human leukocyte antigen-DR in monocytes during septic shock[END_REF]. Moreover, in vitro studies have demonstrated that the simultaneous interference of multiple mechanisms is behind the reduction of MHC II expression in experimental endotoxin tolerance [START_REF] Wolk | Multiple mechanisms of reduced major histocompatibility complex class II expression in endotoxin tolerance[END_REF]. This monocyte deactivation after an acute response to lipopolysaccharide has been proposed as a valuable model of postinflammatory immunodeficiency [START_REF] Wolk | Impaired antigen presentation by human monocytes during endotoxin tolerance[END_REF][START_REF] Docke | Monocyte deactivation in septic patients: Restoration by IFN-gamma treatment[END_REF]. Among the different mechanisms evoked, a marked transcriptional down regulation of the genes involved in the MHC II process was observed, which may be related to the reduced expression of the transcription factor class II transactivator (CIITA) [START_REF] Wolk | Multiple mechanisms of reduced major histocompatibility complex class II expression in endotoxin tolerance[END_REF].

The first requirement of HLA-DR expression on cells is the synthesis of the nonpolymorphic ␣ and highly polymorphic ␤ chains. Then, the biosynthesis and transport of MHC II involves various accessory molecules such as chaperon proteins and enzymes. The genes encoding these major components of the antigen processing and presentation pathway are tightly and coordinately regulated at the level of transcription as illustrated by extensive research performed in patients with bare lymphocyte syndrome, a hereditary immunodeficiency resulting from the absence of MHC II expression [START_REF] Reith | The bare lymphocyte syndrome and the regulation of MHC expression[END_REF].

The aim of our study was to investigate the expression of MHC II genes in septic shock by measuring in whole blood the messenger RNA (mRNA) levels of a gene panel, which is essential to ensure MHC II protein structure, transport, and peptide loading.

MATERIALS AND METHODS

Study Design. Forty-one consecutive patients with septic shock admitted to the surgical or medical intensive care units of a university hospital were enrolled in the study. This prospective study was approved by the local ethics committee, which waived the need for informed consent. Septic shock was defined according to the consensus conference of the American College of Chest Physicians/ Society of Critical Care Medicine [START_REF] Levy | SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference[END_REF]. Severity was assessed using the Simplified Acute Physiology Score (SAPS) II [START_REF] Gall | A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study[END_REF]. Because it is essentially the restoration after day 4 -5 of HLA-DR expression on monocytes that was found to have a good prognostic value [START_REF] Monneret | Analytical requirements for measuring monocytic human lymphocyte antigen DR by flow cytometry: Application to the monitoring of patients with septic shock[END_REF][START_REF] Tschaikowsky | Coincidence of pro-and antiinflammatory response in the early phase of severe sepsis: Longitudinal study of mononuclear histocompatibility leukocyte antigen-DR expression, procalcitonine, C-reactive protein, and changes in T-cell subsets in[END_REF], a first blood sample was collected early after the onset of shock (Ͻ72 hrs) and, when possible, a second one later in the course of the syndrome (Ͼ72 hrs). Mortality was defined as death occurring within 28 days after the onset of septic shock. Fifteen healthy volunteers (mean age, 56 Ϯ 4 yrs; male:female ratio, 1:25), enrolled regularly during the study, served as controls.

Flow Cytometry and DNA Extraction. HLA-DR expression on monocytes was assessed by using flow cytometry (EPICS XL; Beckmann-Coulter) as previously described [START_REF] Monneret | Analytical requirements for measuring monocytic human lymphocyte antigen DR by flow cytometry: Application to the monitoring of patients with septic shock[END_REF]. Genomic DNA was extracted from the cell pellet using a standard phenolic extraction protocol as previously described (21).

RNA Extraction from Purified Monocytes and Whole-Blood Samples. For a random subset of patients (n ϭ 6) and healthy donors (n ϭ 3), monocytes were studied from blood samples collected in ethylenediaminetetraacetic acid tubes. Monocytes were positively isolated (anti-CD14 antibodies, isotype: mouse immunoglobulin-G2a, clone TUK4; Miltenyi Biotec, Bergisch Gladbach, Germany) from peripheral blood mononuclear cells using a magnetic cell separation system (MACS; Miltenyi Biotec) with a cell purity higher than 90%. Total RNA was isolated from the purified monocytes using TRIzol Reagent (Invitrogen, Carlsbad, CA) and treated with DNAse (Ambion, Austin, TX).

The whole-blood mRNA expression profile was studied using blood samples collected directly in PAXgene blood RNA tubes (PreAnalytix, Hilden, Germany). Total RNA was extracted using the PAXgene Blood RNA kit (PreAnalytix). Before RNA elution, the residual genomic DNA was digested using the RNase-Free DNase set (Qiagen, Hilden, Germany).

Total RNA was reverse transcribed into complementary DNA (cDNA) using the ThermoScript reverse transcriptasepolymerase chain reaction system (Invitrogen) according to the manufacturer's instructions. The mRNA expression of genes required for MHC II-restricted antigen presentation (HLA-DRA, -DRB, -DMA, -DMB, invariant chain [Ii], cathepsin S) and CIITA was quantified using q-reverse transcriptase-polymerase chain reaction. PCR reactions were performed in a LightCycler instrument using the Fast-Start DNA Master SYBR Green I real-time PCR kit according to the manufacturer's instructions (Roche Molecular Biochemicals, Basel, Switzerland). Thermocycling was performed in a final volume of 20 L containing 3 mM MgCl 2 and 0.5 M each of the required primers. PCR was performed with an initial denaturation step of 10 mins at 95°C, followed by 40 cycles of a touchdown PCR protocol (10 secs at 95°C, 10 secs annealing at 68 -58°C, and 16 secs extension at 72°C).

The cDNA calibrators were prepared from purified PCR amplicons obtained with the corresponding primers (Table 1). To control the PCR specificity, PCR products were sequenced and matched to the GeneBank database. Expression of the housekeeping gene PPIB encoding for cyclophilin B was investigated using specific cDNA standards and ready-to-use primer mixes obtained from Search-LC (Heidelberg, Germany). The efficiency of using PPIB mRNA levels as a reference for target mRNA quantification in human peripheral blood was previously studied at our institute (25). The Second Derivative Maximum Method was used with the LightCycler software to automatically determine the crossing point for individual samples as previously described (26). Serial dilutions of each cDNA standard were prepared in quadruplicate to generate standard curves. Relative standard curves describing the PCR efficiency of selected genes and PPIB were created and used to perform efficiency-corrected quantification with the LightCycler Relative Quantification Software (Roche Molecular Biochemicals).

mRNA Quantification of HLA-DRB1 Alleles. To take into account the interindividual allelic variation of HLA-DRB1, specific primers (Table 1) were designed according to the patient's DRB1 genotype (HLA-DR oligo-detection kit bioMérieux, Marcy l'Etoile, France) (27) to perform q-reverse transcriptase-polymerase chain reaction. Forward DRB1 primers annealed to the first hypervariable region of exon 2. Reverse primer annealed to a conserved region of DRB genes located between the second and third hypervariable regions of exon 2. In each heterozygous individual, both DRB1 alleles were tested. The overall number of HLA-DRB1 mRNA copies corresponding to both alleles was normalized to the number of PPIB mRNA copies.

Statistical Analysis. The comparison between groups was performed with the nonparametric Mann-Whitney U test. As a result of the lack of serial data for the healthy donors, the groups were compared only once using the earliest measurement for each septic patient, which was obtained during the first 72 hrs after the onset of septic shock.

The nonparametric Wilcoxon's paired test was used to assess time-related individual variation of the parameters between the two observation periods. Among the 41 patients enrolled in the study, nine patients could not be in-cluded in this analysis either because they deceased or left the intensive care unit before the second sample or because the mRNA quantification was not successful for the all-gene panel.

Correlation between parameters was studied with the Spearman's rankcorrelation test. We considered p Ͻ .05 to be significant without correction of the number of tests performed. Box and whisker plots, with representation of the median, 75th and 90th percentiles, and outliers, were used for most of the graphic representation.

RESULTS

Patients. The global mortality rate was 35%. Mean SAPS II admission score was significantly increased in nonsurvivors compared with survivors. None of the other clinical and demographic characteristics described in Table 2 displayed significant differences between the two groups.

Surface Expression of HLA-DR on Monocytes. During the first observation period (first 72 hrs after the onset of septic shock), compared with healthy donors (n ϭ 15), the expression of HLA-DR on monocytes was severely and significantly reduced in septic shock patients (n ϭ 41) without overlap (p Ͻ .001). Pa-tients who survived showed a higher percentage (Fig. 1) and mean fluorescence intensities (data not shown) of HLA-DRpositive monocytes than patients who did not, both in the early (survivors: n ϭ 27 vs. nonsurvivors: n ϭ 14) and late (survivors: n ϭ 24 vs. nonsurvivors: n ϭ 10) phases of septic shock. Comparing the periods of days 1-3 and 4 -10, a significant increase of monocytic HLA-DR expression was observed in survivors (p ϭ .001, Wilcoxon's paired test) but not in nonsurvivors. Regarding the number of circulating monocytes, no significant differences were observed between survivors and nonsurvivors both at the early and late phase of the septic shock, and no significant variations were observed over time in each group (survivor days 1-3: 556 Ϯ 367; nonsurvivors days 1-3: 720 Ϯ 454; survivor days 4 -10: 674 Ϯ 333; nonsurvivors days 4 -10: 881 Ϯ 495 monocytes/L).

HLA-DRB mRNA Level in Purified Monocytes Is Highly Correlated with Surface Expression. The expression of HLA-DRB mRNA, including DRB1 alleles and, for most HLA haplotypes, those of the second DRB locus expressed (i.e., DRB3, DRB4, or DRB5), was measured using DRB generic primers in purified monocytes from six patients with septic shock (three survivors and three nonsurvivors; median SAPS II score: 65; median sampling time: day 6) and three healthy volunteers. After monocyte purification, a fraction of cells was also used to measure the HLA-DR surface expression on monocytes by flow cytometry. The HLA-DRB mRNA expression in purified monocytes was systematically lower in septic shock patients than in healthy volunteers (Fig. 2). Moreover, HLA-DRB mRNA level was correlated both with the percentage (r ϭ .9; p Ͻ .01) and the mean fluorescence intensities (r ϭ .87; p ϭ .01) of HLA-DRpositive monocytes.

HLA-DRB1 and -DRA mRNA Expression in Whole Blood. Based on the HLA-DRB1 genotype determined in patients and controls, DRB1 allele-specific mRNA was quantified in whole-blood samples obtained from a random subset of 11 healthy volunteers and the first 19 septic shock patients (nine survivors and ten nonsurvivors) enrolled prospectively in the study. The overall DRB1 mRNA was significantly decreased in septic shock patients compared with healthy volunteers (p Ͻ .0001) (Fig. 3). When survivors and nonsurvivors were compared, there was no difference in the DRB1 mRNA level in the early phase of septic shock (days 1-3). However, when measured later in the syndrome (days 4 -10), the DRB1 mRNA expression was significantly higher in survivors (p ϭ .02), suggesting an ongoing recovery of the HLA-DRB transcription activity. Moreover, both in the early and late phase of septic shock, we observed a major and significant down regulation of DRA mRNA expression in whole blood compared with control values (p Ͻ .0001) (Fig. 4A). During both observation periods, DRA mRNA expression was higher in the survivor than in the nonsurvivor group. The difference reached statistical significance only at days 4 -10.

Both HLA-DRA and DRB1 mRNA levels measured in whole blood showed a significant correlation with the percentage (HLA-DRA: r ϭ .74, p Ͻ .0001; HLA-DRB1: r ϭ .72, p Ͻ .0001) or mean fluorescence intensities (data not shown) of HLA-DR ϩ monocytes measured by means of flow cytometry.

mRNA Expression of MHC-II Genes, CIITA, and Cathepsin S in Whole Blood. MHC II ␣/␤ heterodimers initially assemble in the endoplasmic reticulum with Ii, which acts as a chaperone to stabilize the heterodimer and targets class II molecules to late endosomal compartments (29). In the present study, we found a decreased expression of Ii mRNA in septic shock (p Ͻ .0001), especially in nonsurvivors (Fig. 4B).

After reaching the endosomal compartment, Ii is partially released from the complex, leaving a residual peptide named class II invariant chain peptide. The generation of class II invariant chain peptide is mediated by cysteine endopeptidases and especially cathepsin S. In our study, we found a reduced expression of cathepsin S mRNA in the overall population of septic shock compared with healthy volunteers (p Ͻ .0001). In the early phase of septic shock, the cathepsin S mRNA level did not differ between patients who died and those who survived, whereas it was significantly increased in survivors in the late observation period (days 4 -10) (Fig. 4C).

The release of class II invariant chain peptide and its replacement with antigenic peptides is catalyzed by HLA-DM (29). Compared with control values, septic shock was characterized by a down regulation of HLA-DM mRNA, especially the HLA-DMA isoform (p Ͻ .001). The expression of both isoforms was significantly lower in nonsurvivor patients (Fig. 4, D andE).

The expression of Ii, HLA-DMA, -DMB, -DRA, and DRB genes are known to be coordinately regulated at the level of transcription by a conserved set of factors, of which CIITA has a central role (29). Here, we found a marked down regulation of CIITA mRNA expression in septic shock compared with control values (p Ͻ .0001), especially in nonsurvivors (Fig. 4F).

The correlation analysis showed a significant correlation between whole-blood HLA-DRA, -DMA, Ii, and CIITA mRNA levels (Table 3). Moreover, HLA-DMA and Regarding the time-course analysis, a significant increase in the whole-blood DRA mRNA level was observed in survivors (Wilcoxon's paired test, p ϭ .001), whereas it remained low and stable in nonsurvivors (Fig. 5). The same trends were observed for HLA-DMA, -DMB, Ii, and CIITA but not for cathepsin S (Table 4).

DISCUSSION

The regulation of genes required for MHC II-restricted antigen presentation plays a fundamental role in the adaptive immune response, because the encoded molecules direct the development, activation, and homeostasis of CD4 ϩ T cells. During sepsis-induced immunoparalysis, the deregulated expression of monocytic HLA-DR seems to mediate a sustained depression of the antigen-presenting cell's functions [START_REF] Manjuck | Decreased response to recall antigens is associated with depressed costimulatory receptor expression in septic critically ill patients[END_REF][START_REF] Wolk | Impaired antigen presentation by human monocytes during endotoxin tolerance[END_REF][START_REF] Docke | Monocyte deactivation in septic patients: Restoration by IFN-gamma treatment[END_REF]. The magnitude of the immunoparalysis is believed to have a major impact in the development of secondary septic complications and thus on the patient's outcome (30,31).

Previous studies have shown that low HLA-DR expression on monocytes from patients with sepsis may a predictive value for a fatal outcome [START_REF] Muller Kobold | Leukocyte activation in sepsis: Correlations with disease state and mortality[END_REF][START_REF] Tschaikowsky | Coincidence of pro-and antiinflammatory response in the early phase of severe sepsis: Longitudinal study of mononuclear histocompatibility leukocyte antigen-DR expression, procalcitonine, C-reactive protein, and changes in T-cell subsets in[END_REF]32). It is essentially the restoration after days 4 -5 of HLA-DR expression on monocytes that was found to have a good prognostic value [START_REF] Monneret | Analytical requirements for measuring monocytic human lymphocyte antigen DR by flow cytometry: Application to the monitoring of patients with septic shock[END_REF][START_REF] Tschaikowsky | Coincidence of pro-and antiinflammatory response in the early phase of severe sepsis: Longitudinal study of mononuclear histocompatibility leukocyte antigen-DR expression, procalcitonine, C-reactive protein, and changes in T-cell subsets in[END_REF]. In accordance, we observed a significant decreased monocytic HLA-DR expression both in survivors and nonsurvivors, with a significant recovery in patients who survived between days 1-3 and 4 -10. However, we observed an increased HLA-DR expression in survivors as compared with nonsurvivors even at the early phase of the syndrome, which is in contrast with the previously published data. A monitoring study of a large cohort of patients is required to clarify this point.

Results obtained on purified monocytes from septic shock patients showed that the decreased expression of HLA-DR could be mainly mediated by reendocytosis and cellular sequestration of MHC II molecules [START_REF] Fumeaux | Role of interleukin-10 in the intracellular sequestration of human leukocyte antigen-DR in monocytes during septic shock[END_REF]. In this study, Fumeaux and Pugin observed only a slight, but not significant, down regulation of HLA-DRA mRNA production. In contrast, we found a significant correlation between the cellsurface expression of HLA-DR and the overall expression of HLA-DRB mRNA in purified monocytes, both in septic shock patients and healthy volunteers. Although our results were based on a small number of samples collected in the late phase of the syndrome, the coordinated transcriptional regulation of both HLA-DRA and -DRB chains [START_REF] Reith | The bare lymphocyte syndrome and the regulation of MHC expression[END_REF] suggested that we could expect results similar to those of Fumeaux and Pugin (15). This discrepancy might be explained both by clinical and technical differences. Indeed, different monocyte purification methods, which could display different in vitro cellstimulation effects and thus have a different impact on the transcriptional resumption, were used. Moreover, although the median age of patients was similar in both studies, Fumeaux and Pugin [START_REF] Fumeaux | Role of interleukin-10 in the intracellular sequestration of human leukocyte antigen-DR in monocytes during septic shock[END_REF] measured mRNA levels in samples collected earlier in the course of the syndrome. It is important to emphasize that Wolk and co-workers ( 16) demonstrated that multiple mechanisms, including a high transcriptional regulation, are involved in the reduction of HLA-DR expression in a model of lipopolysaccharide-primed monocytes. Finally, during the completion of this article, a study describing the effect of cortisol on HLA-DR down regulation reported, by using the same monocyte purification method as us, a decreased expression of HLA-DRA mRNA in a small cohort of nine septic patients when compared with a set of control patients (33).

To get rid of the potential in vitro cell-stimulation effects of the monocyte purification step, we decided to extend our investigations using the PAXgene Blood RNA system. This system provides an efficient and standardized method for whole-blood collection that prevents any postsampling stimulation (34). It is important to emphasize that HLA-DR is not only expressed by circulating monocytes, which is an important limitation of using PAXgene samples. Nevertheless, in agreement with the results obtained on purified monocytes, HLA-DRB1 and -DRA mRNA levels were decreased in whole blood from septic shock patients and showed significant correlation with the monocyte membrane HLA-DR expression. Moreover, we could not take into account the percentage of starting RNA that came from each subpopulation, which is clearly a second limitation of using the PAXgene tubes.

The genes encoding the ␣ and ␤ chains of all MHC II isotypes together with the genes encoding the major components of the antigen processing pathway (invariant chain and HLA-DM) are mainly regulated in a coordinated and concerted fashion by a conserved group of transcription factors [START_REF] Reith | The bare lymphocyte syndrome and the regulation of MHC expression[END_REF]29). This requires the recruitment of the MHCspecific transcriptional coactivator CIITA, which is known as the master regulator of MHC II genes (35, 36). In the present study, whole-blood mRNA levels of HLA-DRA, invariant chain, and HLA-DMA were strongly correlated together giving support to a transcriptional coregulation of these genes both in septic shock patients and healthy volunteers. As already described in multiple tissues, HLA-DRA, -DMA, and invariant chain mRNA levels were highly correlated with the CIITA mRNA expression, illustrating CIITA as the rate-limiting factor of MHC II gene expression (37). Taken together, these observations would suggest that the transcriptional regulation of CIITA is involved in the reduction of MHC II gene expression observed in the course of septic shock and is implicated in the transcriptional reinitialization observed in survivor patients.

Cathepsin S contributes to late stages of Ii degradation in antigen-presenting cells and thus allows subsequent MHC II peptide loading (38, 39). Cathepsin S transcription does not depend on CIITA regulation. In agreement, no correlation was observed between the whole-blood mRNA level of cathepsin S and CIITA or other genes required for MHC II-restricted antigen presentation. The down regulation of cathepsin S mRNA level observed in our cohort of septic shock patients and in lipopolysaccharide-primed 16) is an illustration that CI-ITA-independent transcriptional machinery is deactivated during immunoparalysis. The lack of significant time-course progression between days 1-3 and 4 -10 observed in our study suggests that the transcriptional resumption of cathepsin S has a secondary role in the MHC II recovery observed in patients who survived.

The decreased expression of monocyte membrane MHC II observed in septic shock patients is at least partly mediated by interleukin-10 (15). However, interleukin-10-induced reduction of MHC II seems to be solely the result of reendocytosis and intracellular sequestration of mature MHC II molecules with no effect on MHC II transcription [START_REF] Fumeaux | Role of interleukin-10 in the intracellular sequestration of human leukocyte antigen-DR in monocytes during septic shock[END_REF]40). Transforming growth factor-␤ 1, catecholamine, and prostaglandin E2 are other major antiinflammatory mediators identified in critically ill patients. Transforming growth factor-␤ 1 has been demonstrated in vitro to down regulate MHC II mRNA expression through the suppression of CIITA mRNA transcription (41, 42), whereas prostaglandin E 2 suppresses MHC II mRNA expression in macrophages (43). These observations, together with our results, suggest that the down regulation of genes required for MHC IIrestricted antigen presentation observed in septic shock might be mediated by a panel of antiinflammatory mediators that remains to be described.

CONCLUSIONS

By analyzing whole blood as well as isolated monocytes from septic shock pa-tients, we described a global transcriptional down regulation of a panel of genes required for MHC II-restricted antigen presentation. Although no direct conclusion on the mechanism taking place in the monocytes can be drawn from our PAXgene whole-blood results, we showed that the decreased surface expression of HLA-DR on monocytes was associated with the down regulation of HLA-DRA, DRB, and, to a lesser extent, with HLA-DMA and Ii mRNA levels in whole blood. This might be the result of the inhibition of various transcriptional mechanisms, of which a shutdown of CIITA mRNA expression should be crucial. In our study, although survivor and nonsurvivor patients were hardly distinguishable in the early phase of the syndrome, the timerelated variation of whole-blood MHC II mRNA levels was different according to the outcome. The transcriptional resumption of CIITA-regulated genes might contribute to the recovery of membrane HLA-DR expression observed in survivor patients. These data obtained mainly from PAXgene whole-blood samples have now to be confirmed by other approaches. Defining the factors that regulate the transcription of MHC II genes will greatly contribute to a better understanding of the immunoparalysis observed in the course of sepsis.

B

  transcription factor class II transactivator (CIITA), and the enzymes involved in the peptide loading cathepsin S, HLA-DMA, and -DMB. The monocyte surface expression of HLA-DR measured by flow cytometry was significantly correlated with the whole-blood mRNA levels of HLA-DRB1 and -DRA and to a lesser extent with the other CIITAregulated genes HLA-DMA and invariant chain. The correlation between HLA-DRB mRNA and cell-surface expression levels was also observed in a small subset of purified monocyte samples. Regarding the temporal relationship, a significant increase of whole-blood HLA-DRA, -DMA, -DMB, invariant chain, and CIITA mRNA level was observed in survivors (p ؍ .001), whereas expressions remained low in nonsurvivors. Conclusions: A global transcriptional down regulation of a gene panel required for MHC II-restricted antigen presentation may occur in the course of septic shock. Our results suggest that the transcriptional resumption of CIITA-regulated genes might contribute to the recovery of membrane HLA-DR expression observed in survivors. These results obtained at the mRNA level support previous reports describing the loss of monocyte HLA-DR at the protein level and thus confirm the potential of measuring monocyte HLA-DR in septic patient followup. (Crit Care Med 2005; 33:31-38) KEY WORDS: human leukocyte antigen-DR; sepsis; blood; messenger RNA; reverse transcriptase-polymerase chain reaction; class II transactivator sis-induced immunoparalysis

Figure 1 .

 1 Figure 1. Percentage of human leukocyte antigen (HLA)-DR-positive monocytes in peripheral blood of survivor (S; n ϭ 27), nonsurvivor (NS; ϭ 14) septic shock patients and healthy volunteers (n ϭ 15) measured by flow cytometry. Septic shock patients were analyzed during the first 72 hrs after the onset of shock and later between days 4 and 10. **p Ͻ .01 vs. survivors, Mann-Whitney U test.

Figure 2 .

 2 Figure 2. Cell surface expression of human leukocyte antigen (HLA)-DR measured by flow cytometry (bars) and HLA-DRB messenger RNA (mRNA) level measured by q-reverse transcriptase-polymerase chain reaction (line) in purified monocytes from healthy volunteers (n ϭ 3) and patients with septic shock (n ϭ 6). The HLA-DRB mRNA level was normalized to that of the housekeeping gene PPIB. The cell surface HLA-DR expression corresponds to the percentage of HLA-DR ϩ monocytes.

Figure 3 .

 3 Figure 3. Human leukocyte antigen (HLA)-DRB1 messenger RNA (mRNA) level in peripheral blood of survivors (S; n ϭ 9) and nonsurvivor (NS; n ϭ 10) septic shock patients and healthy volunteers (n ϭ 11) measured by q-reverse transcriptasepolymerase chain reaction. Primers were selected according to the HLA-DRB1 genotype. Overall HLA-DRB1 mRNA level, corresponding to both alleles, was normalized to that of the housekeeping gene PPIB. Septic shock patients were analyzed during the first 72 hrs after the onset of shock and later between days 4 and 10. *p Ͻ .05 vs. survivors, Mann-Whitney U test.

  Ii mRNA levels showed a trend toward a correlation with the percentage (HLA-DMA: r ϭ .70, p Ͻ .0001; Ii: r ϭ .65, p Ͻ .001) or the mean fluorescence intensities (data not shown) of HLA-DR ϩ monocytes measured by means of flow cytometry.

Figure 4 .

 4 Figure 4. Messenger RNA (mRNA) level of human leukocyte antigen (HLA)-DRA (A), invariant chain (B), cathepsin S (C), HLA-DMA (D), -DMB (E), and class II transactivator (F) in peripheral blood of survivor (S; n ϭ 27) and nonsurvivor (NS; n ϭ 14) septic shock patients and healthy volunteers (n ϭ 15) measured by q-reverse transcriptase-polymerase chain reaction. The mRNA level was normalized to that of the housekeeping gene PPIB. Septic shock patients were analyzed during the first 72 hrs after the onset of shock and later between days 4 and 10. *p Ͻ .05 vs. survivors; **p Ͻ .01 vs. survivors, Mann-Whitney U test.

Figure 5 .

 5 Figure 5. Time-related variation of human leukocyte antigen (HLA)-DRA messenger RNA (mRNA) level in peripheral blood. The mRNA level was measured by q-reverse transcriptase-polymerase chain reaction and normalized to that of the housekeeping gene PPIB. Individual values for survivors (n ϭ 22) and nonsurvivors (n ϭ 10) were taken into account when paired samples were available for the two time periods studied: days 1-3 and 4 -10.

Table 1 .

 1 Design of primers used for the messenger RNA quantification of transcription factor class II transactivator (CIITA) and genes required for major histocompatibility complex II-restricted antigen presentation using real-time polymerase chain reaction

	Gene	Accession No.	Sequence
	HLA-DRA	NM_019111	5Ј-GCCAACCTGGAAATCATGACA-3Ј (sense) (22)
			5Ј-AGGGCTGTTCGTGAGCACA-3Ј (antisense) (22)
	CIITA	NM_000246	5Ј-CCTGCTGTTCGGGACCTAAA-3Ј (sense) (22)
			5Ј-GGATCCGCACCAGTTTGG-3Ј (antisense) (22)
	Invariant chain	NM_004355	5Ј-GCAGAGGCGGTCTTCAACA-3Ј (sense) (15)
			5Ј-GCACCAGGTCTCATGGGAT-3Ј (antisense)
	Cathepsin S	NM_004079	5Ј-CAATAAAGGCCCAGTGTCTGTTG-3Ј (sense) (15)
			5Ј-GATGGTTCATAGTAGACACCACTTCTG-3Ј (antisense) (15)
	HLA-DMA	NM_006120	5Ј-CTGTGTGGCAAGAAGGTATGGGT-3Ј (sense) (23)
			5Ј-CATTGGAGTAGGAGCTTCAGGGA-3Ј (antisense)
	HLA-DMB	NM_002118	5Ј-GGCATCTTTACAGAGCAGAGCAT-3Ј (sense) (23)
			5Ј-ATGTGAAATCCTTTGGAGTCCCA-3Ј (antisense) (23)
	HLA-DRB1*01		5Ј-GGCAGCTTAAGTTTGAATG-3Ј (sense) (24)
			5Ј-AGGCCGCCCCAGCTCCGT-3Ј (antisense) (24)
	HLA-DRB1*03, *08, *11, *12, *13, *14		5Ј-AGTACTCTACGTCTGAGTG-3Ј (sense) (24)
			5Ј-AGGCCGCCCCAGCTCCGT-3Ј (antisense) (24)
	HLA-DRB1*04		5Ј-AGCAGGTTAAACATGAGTG-3Ј (sense) (24)
			5Ј-AGGCCGCCCCAGCTCCGT-3Ј (antisense) (24)
	HLA-DRB1*07		5Ј-CTGTGGCAGGGTAAGTATA-3Ј (sense)
			5Ј-AGGCCGCCCCAGCTCCGT-3Ј (antisense)
	HLA-DRB1*08, *11, *12, *14		5Ј-AGTACTCTACGGGTGAGTG-3Ј (sense)
			5Ј-AGGCCGCCCCAGCTCCGT-3Ј (antisense)
	HLA-DRB1*15, *16		5Ј-CTAAGAGGGAGTGTCATTT-3Ј (sense)
			5Ј-AGGCCGCCCCAGCTCCGT-3Ј (antisense)
	HLA-DRB		5Ј-AGTACCGGGCGGTGACGGAG-3Ј (sense)
			5Ј-TCGCCGCTGCACTGTGAAG-3Ј (antisense)
	HLA, human leukocyte antigen.

Table 2 .

 2 Demographic, clinical characteristics, microbiology, and type of infection

	Survivors	Nonsurvivors	Total
	n ϭ 27 (%)	n ϭ 14 (%)	n ϭ 41 (%)	p

a Median (Q1-Q3).

Table 3 .

 3 Spearman's correlation coefficients between messenger RNA expression of genes required for major histocompatibility complex II-restricted antigen presentation and the transcription factor class II transactivator (CIITA) in peripheral blood

		Invariant
		Chain	HLA-DMA	HLA-DMB	CIITA	Cathepsin S
	HLA-DRA	0.91 a	0.91 a	0.67 a	0.80 a	0.54 a
	Invariant chain	NA	0.92 a	0.64 a	0.73 a	0.44 a
	HLA-DMA	0.92 a	NA	0.64 a	0.83 a	0.46 a
	HLA-DMB	0.64 a	0.64 a	NA	0.53 a	0.37 b
	CIITA	0.73 a	0.83 a	0.53 a	NA	0.57 a
	Cathepsin S	0.44 a	0.46 a	0.37 b	0.57

a NA HLA, human leukocyte antigen; NA, not applicable. a p Ͻ .0001; b p Ͻ .001.

Table 4 .

 4 Wilcoxon's paired test comparing the progression of messenger RNA level for the transcription factor class II transactivator (CIITA) and genes required for major histocompatibility complex II-restricted antigen presentation in septic shock patients between days 1-3 and 4 -10

				Cathepsin	Invariant
		DRA	CIITA	S	chain	DMA	DMB
	Survivors (n ϭ 22)	p Ͻ .01	p Ͻ .01	p ϭ .21	p Ͻ .01	p Ͻ .01	p Ͻ .01
	Nonsurvivors (n ϭ 10)	p ϭ .17	p ϭ .066	p ϭ .85	p ϭ .14	p ϭ .70	p ϭ .44

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ACKNOWLEDGMENTS

We thank Dr. J. M. Tiercy and Dr. W. Reith for assistance with the preparation of the manuscript.