Mice carrying a humanized Foxp2 knock-in allele show region-specific shifts of striatal Foxp2 expression levels.

Genetic and clinical studies of speech and language disorders are providing starting points to unravel underlying neurobiological mechanisms. The gene encoding the transcription factor FOXP2 has been the first example of a gene involved in the development and evolution of this human-specific trait. A number of autosomal-dominant FOXP2 mutations are associated with developmental speech and language deficits indicating that gene dosage plays an important role in the disorder. Comparative genomics studies suggest that two human-specific amino acid substitutions in FOXP2 might have been positively selected during human evolution. A knock-in mouse model carrying these two amino acid changes in the endogenous mouse Foxp2 gene (Foxp2hum/hum) shows profound changes in striatum-dependent behaviour and neurophysiology, supporting a functional role for these changes. However, how this affects Foxp2 expression patterns in different striatal regions and compartments has not been assessed. Here, we characterized Foxp2 protein expression patterns in adult striatal tissue in Foxp2hum/hum mice. Consistent with prior reports in wildtype mice, we find that striatal neurons in Foxp2hum/hum mice and wildtype littermates express Foxp2 in a range from low to high levels. However, we observe a shift towards more cells with higher Foxp2 expression levels in Foxp2hum/hum mice, significantly depending on the striatal region and the compartment. As potential behavioural readout of these shifts in Foxp2 levels across striatal neurons, we employed a morphine sensitization assay. While we did not detect differences in morphine-induced hyperlocomotion during acute treatment, there was an attenuated hyperlocomotion plateau during sensitization in Foxp2hum/hum mice. Taken together, these results suggest that the humanized Foxp2 allele in a mouse background is associated with a shift in striatal Foxp2 protein expression pattern.

Cellular prion protein co-localizes with nAChR beta4 subunit in brain and gastrointestinal tract.

PrP(C), the cellular isoform of prion protein, is widely expressed in most tissues, including brain, muscle and gastrointestinal tract. Despite its involvement in several bioprocesses, PrP has still no apparent physiological role. During propagation of transmissible spongiform encephalopathies (TSE), prion protein is converted to the pathological isoform, PrP(Sc), in a process believed to be mediated by unknown host factors. The identification of proteins associated with PrP may provide information about both the biology of prions and the pathogenesis of TSE. Thus far, PrP(C) has been shown to interact with synaptic proteins, components of the cytoskeleton and intracellular proteins involved in signalling pathways. Here, we describe the association of PrP with the beta4 subunit of nicotinic acetylcholine receptor (nAChR), as indicated by co-immunoprecipitation assays and double-label immunofluorescence. The interaction between prion protein and native beta4 subunit was further studied by affinity chromatography, using immobilized and refolded recombinant PrP as a bait and brain homogenates from normal individuals. Additionally, the participation of beta4 subunit in the pathogenesis of TSE was studied by in vivo assays. beta4(-/-) and wild-type mice were challenged with the RML (Rocky Mountain Laboratories) infectious agent. Transgenic animals displayed altered incubation times but the deletion of beta4 subunit did not result in a significant change of the incubation period of the disease. Our results suggest that PrP(C) is a member of a multiprotein membrane complex participating in the formation and function of alpha3beta4 nAChR.

Gram-positive and gram-negative bacteria do not trigger monocytic cytokine production through similar intracellular pathways.

Toll-like receptors (TLRs) are involved in human monocyte activation by lipopolysaccharide (LPS) and Staphylococcus aureus Cowan (SAC), suggesting that gram-positive and gram-negative bacteria may trigger similar intracellular events. Treatment with specific kinase inhibitors prior to cell stimulation dramatically decreased LPS-induced cytokine production. Blocking of the p38 pathway prior to LPS stimulation decreased interleukin-1alpha (IL-1alpha), IL-1ra, and tumor necrosis factor alpha (TNF-alpha) production, whereas blocking of the ERK1/2 pathways inhibited IL-1alpha, IL-1beta, and IL-1ra but not TNF-alpha production. When cells were stimulated by SAC, inhibition of the p38 pathway did not affect cytokine production, whereas only IL-1alpha production was decreased in the presence of ERK kinase inhibitor. We also demonstrated that although LPS and SAC have been shown to bind to CD14 before transmitting signals to TLR4 and TLR2, respectively, internalization of CD14 occurred only in monocytes triggered by LPS. Pretreatment of the cells with SB203580, U0126, or a mixture of both inhibitors did not affect internalization of CD14. Altogether, these results suggest that TLR2 signaling does not involve p38 mitogen-activated protein kinase signaling pathways, indicating that divergent pathways are triggered by gram-positive and gram-negative bacteria, thereby inducing cytokine production.

Inflammation is probably not a prerequisite for renal interstitial fibrosis in normoglycemic obese rats.

We examined the role of inflammation in the development of renal interstitial fibrosis in Zucker obese rats, which rapidly present kidney lesions in the absence of hypertension and hyperglycemia. Type I and III collagens were quantified using a polarized light and computer-assisted image analyzer. The expression of mRNA encoding matrix components, adhesion molecules, chemokines, and growth factors was followed by RT-PCR. The presence of synthesized proteins as well as lymphocytes and macrophages was determined by immunohistochemistry. Interstitial fibrosis developed in two phases. The first phase occurred as early as 3 mo and resulted from a neosynthesis of type III collagen and fibronectin and a reduction of extracellular matrix catabolism, in parallel with an overexpression of transforming growth factor-beta(1) and in the absence of any lymphocyte or macrophage infiltration. After 6 mo, interstitial fibrosis worsened with a large accumulation of type I collagen, concomitantly with a large macrophage infiltration. Thus inflammation cannot explain the onset of interstitial fibrosis that developed in young, insulinoresistant, normoglycemic, obese Zucker rats but aggravated this process afterward.

A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts.

Because G-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1-EGFP receptor in a dose-dependent fashion (EC(50) = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1-EGFP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs.

Two distinct pathways leading to nuclear apoptosis.

Apaf-1(-/-) or caspase-3(-/-) cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1(-/-) or caspase-3(-/-) cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralization of AIF and CAD is required to suppress the nuclear DNA loss caused by cytoplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletion alone suffices to suppress the nuclear DNA loss contained in extracts from apoptotic Apaf-1(-/-) or caspase-3(-/-) cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptosis. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation. The other pathway, which is caspase-independent, involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin condensation.

Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis.

Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale ( approximately 50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z-VAD. fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a 'necrotic' phenotype. Both in the presence of Z-VAD. fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large-scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase-independent mitochondrial death effector responsible for partial chromatinolysis.

Mass spectrometric identification of proteins released from mitochondria undergoing permeability transition.

Mitochondrial membrane permeabilization is a rate-limiting step of cell death. This process is, at least in part, mediated by opening of the permeability transition pore complex (PTPC) Several soluble proteins from the mitochondrial intermembrane space and matrix are involved in the activation of catabolic hydrolases including caspases and nucleases. We therefore investigated the composition of a mixture of proteins released from purified mitochondria upon PTPC opening. This mixture was subjected to a novel proteomics/mass spectrometric approach designed to identify a maximum of peptides. Peptides from a total of 79 known proteins or genes were identified. In addition, 21 matches with expressed sequence tags (EST) were obtained. Among the known proteins, several may have indirect or direct pro-apoptotic properties. Thus endozepine, a ligand of the peripheral benzodiazepin receptor (whose occupation may facilitate mitochondrial membrane permeabilization), was found among the released proteins. Several proteins involved in protein import were also released, namely the so-called X-linked deafness dystonia protein (DDP) and the glucose regulated protein 75 (grb75), meaning that protein import may become irreversibly disrupted in mitochondria of apoptotic cells. In addition, a number of catabolic enzymes are detected: arginase 1 (which degrades arginine), sulfite oxidase (which degrades sulfur amino acids), and epoxide hydrolase. Although the functional impact of each of these proteins on apoptosis remains elusive, the present data bank of mitochondrial proteins released upon PTPC opening should help further elucidation of the death process.

The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore.

Viral protein R (Vpr) encoded by HIV-1 is a facultative inducer of apoptosis. When added to intact cells or purified mitochondria, micromolar and submicromolar doses of synthetic Vpr cause a rapid dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), as well as the mitochondrial release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor. The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr. Both mitochondrial and cytotoxic Vpr effects are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC). Coincubation of purified organelles revealed that nuclear apoptosis is only induced by Vpr when mitochondria are present yet can be abolished by PTPC inhibitors. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax. Again, this effect is prevented by addition of recombinant Bcl-2. The Vpr COOH terminus binds purified ANT, as well as a molecular complex containing ANT and the voltage-dependent anion channel (VDAC), another PTPC component. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than control cells yet recover Vpr sensitivity when retransfected with yeast ANT or human VDAC. Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

Polarized expression of different monocarboxylate transporters in rat medullary thick limbs of Henle.

Extracellular lactic acid is a major fuel for the mammalian medullary thick ascending limb (MTAL), whereas under anoxic conditions, this nephron segment generates a large amount of lactic acid, which needs to be excreted. We therefore evaluated, at both the functional and molecular levels, the possible presence of monocarboxylate transporters in basolateral (BLMVs) and luminal (LMVs) membrane vesicles isolated from rat MTALs. Imposing an inward H(+) gradient induced the transient uphill accumulation of L-[(14)C]lactate in both types of vesicles. However, whereas the pH gradient-stimulated uptake of L-[(14)C]lactate in BLMVs was inhibited by anion transport blockers such as alpha-cyano-4-hydroxycinnamate, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), and furosemide, it was unaffected by these agents in LMVs, indicating the presence of a L-lactate/H(+) cotransporter in BLMVs, but not in LMVs. Under non-pH gradient conditions, however, the uptake of L-[(14)C]lactate in LMVs was transstimulated 100% by L-lactate, but by only 30% by D-lactate. Furthermore, this L-lactate self-exchange was markedly inhibited by alpha-cyano-4-hydroxycinnamate and DIDS and almost completely by 1 mM furosemide, findings consistent with the existence of a stereospecific carrier-mediated lactate transport system in LMVs. Using immunofluorescence confocal microscopy and immunoblotting, the monocarboxylate transporter (MCT)-2 isoform was shown to be specifically expressed on the basolateral domain of the rat MTAL, whereas the MCT1 isoform could not be detected in this nephron segment. This study thus demonstrates the presence of different monocarboxylate transporters in rat MTALs; the basolateral H(+)/L-lactate cotransporter (MCT2) and the luminal H(+)-independent organic anion exchanger are adapted to play distinct roles in the transport of monocarboxylates in MTALs.

Application of confocal scanning laser microscopy to 3-D DNA image cytometry of prostatic lesions.

OBJECTIVE: To explore the potential of three-dimensional (3-D) image cytometry for the measurement of DNA content in prostatic specimens using confocal scanning laser microscopy (CSLM) and 3-D image analysis. STUDY DESIGN: Thick tissue slices (100 microns), stained for DNA with chromomycin A3, from four patients with benign hyperplasia, prostatic intraepithelial neoplasia (PIN), well- and poorly differentiated adenocarcinoma of the prostate, were studied. Two different blocks from the same slice were studied for each case. Cell nuclei were segmented automatically. DNA content and nuclear volume were measured. RESULTS: DNA histograms showed a single peak in the diploid range for the hyperplasia and PIN cases. For the case of well-differentiated carcinoma, two peaks were observed, one in the diploid range and one in the tetraploid range. The two peaks were observed on two different blocks of the same tissue slice. Poorly differentiated carcinoma was characterized by an aneuploid distribution. For the cases of PIN and carcinoma, we observed a considerable variation in nuclear volume. CONCLUSION: The results indicate the potential of 3-D image cytometry for the measurement of DNA content in prostatic specimens while preserving tissue architecture.

Spatial distribution of cytoskeleton intermediate filaments during fetal rat hepatocyte differentiation.

The construction of the liver parenchyma throughout fetal development depends on the elaboration of intercellular contacts between epithelial cells and between epithelial and mesenchymal cells. During this time, the spatial distribution of cytokeratins in hepatocytes shows a striking evolution as demonstrated by confocal microscopy and image analysis. In the early stages of fetal rat development, the liver is mainly a hematopoietic organ and hepatocytes represent fewer than 40% of all liver cells. At this time, cytokeratin filaments are scarce and are randomly distributed inside the cytoplasm. A coexpression of desmin and cytokeratin is found in some cells. Intercellular contacts between epithelial and mesenchymal cells are more numerous than between epithelial cells. Later in development, hepatocytes are arranged in a "muralium duplex" architecture (two-cell-thick sheets). Contacts between hepatocytes become more numerous and bile canaliculi become well developed. The density of cytokeratin filaments increases and appears to be very high near the bile canaliculi. In adult liver, hepatocytes are arranged in a "muralium simplex" architecture. Cytokeratin filaments show a symmetrical distribution in relation to the nuclear region. The highest density of filaments is found near the cytoplasmic membrane. Variations of the spatial distribution of intermediate filaments throughout hepatocyte differentiation were investigated in a pilot study using computerized image analysis. We found significant differences between the filament networks in fetal and adult hepatocytes.

Three-dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks of prostatic lesions.

DNA ploidy provides important information for the evaluation of the prognosis of prostate cancer. For the purpose of DNA cytometry and nuclear measurements, we developed an image processing system for the acquisition and processing of three-dimensional (3D) images based on confocal scanning laser microscopy (CSLM). The advantage of the CSLM is the preservation of the tissue architecture and the possibility of multilabeling. It is possible to determine both individual nuclear features and cellular features and the degree of the spatial heterogeneity of several markers. Special attention was paid to the development of the automatic method for the 3D segmentation of cell nuclei. Thick tissue slides (100 microm), stained for DNA with chromomycin A3, from 4 patients (with benign hyperplasia, prostatic intraepithelial neoplasia (PIN), and well-and poorly-differentiated adenocarcinoma of the prostate), were studied in order to test the practicability of the developed methodology. DNA histograms showed a single peak in the diploid range for the hyperplasia and PIN cases. For the case of well-differentiated carcinoma, 2 peaks were observed, 1 in the diploid range and I in the tetraploid range. The case of poorly-differentiated carcinoma was characterized by an aneuploid distribution. For the cases of PIN and carcinomas, we observed a considerable variation of the volume of nuclei.

Application of confocal scanning laser microscopy for an automated nuclear grading of prostate lesions in three dimensions.

Confocal scanning laser microscopy provides the opportunity to obtain three-dimensional (3-D) images by piling up consecutive confocal planes. This technique was applied to capture 3-D images from 100-microns-thick tissue blocks from prostate lesions (hyperplasia, dysplasia, adenocarcinomas). Automated methods were implemented to perform a nuclear grading of 3-D cell nuclei from these specimens. Special attention was focused on the development of a new approach to 3-D chromatin texture analysis. This method uses mathematical morphology operations to tessellate the chromatin into homogeneous domains. The nuclear features (volume, shape, texture) were subjected to a discriminant analysis. Using a set of five features, the classification of cell nuclei yielded an accuracy of 96.3%. The results indicate the potential of 3-D imaging and analysis techniques for an automated nuclear grading of prostate lesions.

Quantitative image analysis of cytokeratin filament distribution during fetal rat liver development.

Recent reports suggest that not only chemical but also mechanical influences from the cellular environment could have profound effects on gene activity and could act on cell differentiation and proliferation. These mechanisms involve a tissue matrix system, which includes extracellular matrix, nuclear matrix, and cytoskeleton. Supposing that the cytoskeleton mediates mechanical transduction from the cell environment to the nucleus, significant differences in the spatial distribution of the cytoskeleton network could reflect variations in environmental signals. Hepatocytes during fetal development provide a useful model to study such variations, because a progressive establishment of intercellular contact is reported. The aim of this work is to discriminate steps in hepatocyte differentiation from fetal to adult livers, using computerized quantitative image analysis of cytokeratin (C8) immunofluorescent localization, visualized by confocal scanning laser microscopy. The filament structure is represented by the gray-scale skeleton of the digital images obtained by specially designed segmentation methods. A set of line features was investigated, including number and length of lines, orientation of lines, and the fractal dimension of the filament network. The features studied showed highly significant differences throughout liver development, with an increase of the total amount of cytokeratin filaments. We could also demonstrate a modification in the structure of the network, being more and more dense, with an increase of connecting points. Moreover, it has been shown that filaments have some orientation in fetal hepatocytes, and that directionality. Thus, significant differences in the pattern of the cytokeratin filament network according to the stages of hepatocyte differentiation have been demonstrated with objective and quantitative methods.

A dual approach to structural texture analysis in microscopic cell images.

The computer-based quantitative analysis of microscopic cell images provides important diagnostic information in clinical and experimental pathology. The arrangement of various cell structures can be described as texture. We developed a new approach to structural texture analysis. It assumes that texture consists of homogeneous regions (texture primitives). Texture can be regarded in a dual way--as a composition of regions or as a pattern composed of the regions' boundaries (lines). We implemented methods for the segmentation of regions and lines in grayscale images. The detection of regions is followed by a region-growing process to avoid an oversegmentation. The segmented regions and lines are stored in a uniform data structure which reflects their arrangement in the image. The presented methods were applied to study the chromatin distribution in cell nuclei and the development and differentiation of intermediate filaments in fetal liver cells.

Chromatin texture analysis in three-dimensional images from confocal scanning laser microscopy.

OBJECTIVE: Three-dimensional (3D) images of tissue blocs can be obtained through optical sectioning by a confocal scanning laser microscope and by piling up of consecutive confocal planes. These images offer the opportunity to quantitate biologic structures (i.e., nuclear chromatin) in 3D. Chromatin texture analysis is regarded as a key element in nuclear grading of malignant lesions. The present study introduced a new method of structural texture analysis of 3D chromatin distribution in cell nuclei. The method was assessed for its validity in a pilot study. STUDY DESIGN: The study group consisted of 300 3D cell nuclei from prostatic lesions, 100 each of hyperplasia, dysplasia and well-differentiated carcinoma. A set of 15 texture features was studied for its classification capability in a discriminant analysis. RESULTS: A classifier that confined three texture features yielded a classification accuracy of 91.7% for the training set (60 nuclei). The classification accuracy for the test set (240 nuclei) was 93.3%. CONCLUSION: The new method demonstrated its potential for 3D chromatin analysis. Further studies on large numbers of cases are necessary to evaluate the use of 3D nuclear grading for diagnostic pathology.

Toward objective prognostic grading of prostatic carcinoma using image analysis.

At present there is no generally accepted histopathologic grading system for prostatic carcinoma that would be reproducible, and it is still unclear which system provides the most reliable individual prognosis. The objective of this study was to evaluate the relationship between morphometric features and prognosis in patients with clinical stage B prostatic carcinoma using computerized image analysis. Standardized histologic, Feulgen-stained slides from 23 patients operated on for clinical stage B prostatic carcinoma were studied. Dimension-, form- and texture-related nuclear features, both classic and fractal, were computed for at least 100 nuclei per slide. Patients were divided into low-risk and high-risk groups, using as criteria the absence or presence of metastases, respectively, three years after the operation. A discriminant function based on five chromatin texture-related features allowed complete separation between groups with a good or poor prognosis. These preliminary results, however, need to be confirmed on a larger set of patients.

Towards an objective prognostic index of acute graft-versus-host disease.

Acute graft-versus-host disease (AGVHD) is one of the major complications of allogenic non-T-depleted HLA-compatible bone marrow transplantation. It is not yet possible to predict the clinical evolution of the disease at the time of its first manifestation. Twenty patients who initiated the disease with only moderate skin involvement were selected consecutively among those followed between January 1985 and December 1988 in the Bone Marrow Transplantation Unit, Saint Louis Hospital, Paris. A skin biopsy was performed at the onset of the AGVHD for each patient. For each biopsy, one 5-microns thick section was fixed by Bouin's solution, stained with hematoxylin and eosin and studied by image analysis at a final calibration of 7.6 pixels/microns. Ten patients did not exceed grade I (low risk group) and ten developed a more severe grade (high risk group) in the evolution of the disease. The mean and coefficients of variation, skewness and kurtosis of dimension-, form- and texture-related parameters of the nuclei of lymphocytes infiltrating the skin were investigated for their ability to discriminate between the high and low risk groups. The best discrimination was obtained using texture-related variables. An index containing 5 texture-related variables gave the maximum separation between the two groups, with a 100% correct classification. Our results represent a learning-step towards the development of a prognostic index of AGVHD.