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Sialoblastoma is a very rare congenital salivary gland tumor. No consensus has been reached concerning the treatment of this tumor due to its rarity. The treatment of reference is surgery, which can be mutilating, in the case of a locally invasive tumor. The treatment of metastatic disease is also controversial. The authors report a new case of a 6-year-old girl with a progressively growing left parotid mass since birth. The first cytological diagnosis was that of pleomorphic adenoma. Due to local progression, superficial parotidectomy was performed at the age of 3.5 years and revealed a diagnosis of sialoblastoma. Six months later, local recurrence and lung metastasis were treated by neoadjuvant chemotherapy with a very good partial response on the local recurrence and the lung metastasis, allowing complete parotidectomy with sacrifice of the facial nerve. Bilateral lung biopsies after adjuvant chemotherapy showed total necrosis. No recurrence was observed with a follow-up of 1 year. This case and a review of the literature confirm the very good chemosensitivity of this tumor and argue in favor of neoadjuvant chemotherapy for locally invasive tumors rather than extensive mutilating surgery.

Resume La maladie de Hirschsprung est la plus frequente des etiologies d’occlusion intestinale chez l’enfant. Cette maladie est caracterisee par l’absence de cellules ganglionnaires dans la sous-muqueuse et dans les plexus myenteriques du tube digestif a sa partie distale. A cote de la forme classique rectosigmoidienne (80 % des cas) sont decrites une forme colique totale (10 %), une forme etendue (1 %) et une forme courte rectale (9 %). Cette anomalie se traduit par une dilatation au-dessus de la zone pathologique. Actuellement, les formes sporadiques comme les formes familiales font l’objet d’etudes genetiques, avec la decouverte de trois regions chromosomiques connues pour etre liees a la maladie de Hirschsprung. L’expression clinique de cette maladie debute a la naissance avec un syndrome occlusif ou plus tardivement par une constipation opiniâtre du nourrisson. La complication principale est l’enterocolite aigue qui en fait toute la gravite et peut imposer une derivation intestinale en urgence. Le diagnostic de la maladie est a la fois clinique, radiologique et surtout anatomopathologique. En effet, c’est la biopsie rectale qui affirme le diagnostic en mettant en evidence l’association d’absence de cellules ganglionnaires et l’hypertrophie des filets nerveux. La prise en charge des enfants atteints de maladie de Hirschsprung a considerablement evolue ces dernieres annees, la chirurgie en un temps, sans colostomie initiale, devient a la fois le traitement de choix de la maladie de Hirschsprung rectosigmoidienne, mais aussi de plus en plus souvent de la maladie de Hirschsprung totale.

We have previously shown that the human developing pancreas, as a tissue under construction and remodeling, is composed of epithelial ducts and differentiated endocrine cells surrounded by mesenchyme. The physiologic importance of resident tissue leukocytes, expected to enter through the mesenchyme in remodeling functions, prompted us to investigate human developing pancreases for the presence of leukocyte lineages and for expression of cytokines and receptors involved in their recruitment and differentiation. Immunohistochemistry studies were performed on 69 human, paraffin-embedded pancreases at 6–12 weeks of development (WD). Cytokines and receptor transcripts were monitored by reverse transcription (RT)–PCR, by immunohistochemistry when antibodies were available or by in situ hybridization (ISH). We show that numerous cells expressing CD45RA, HLADR and CD68 antigens are cellular components of the mesenchyme of all the pancreases at 6–12 WD. So-called constitutive chemokines (SLC (CCL19), stromal-derived factor 1 (SDF1) (CXCL12)) and a macrophage-specific growth/survival factor, colony-stimulating factor 1 (CSF1), were detected in epithelial duct cells. Both epithelial and mesenchymal cells expressed chemokine receptors, suggesting a role in leukocyte recruitment and possibly in early pancreatic development. In conclusion, we demonstrated the presence of CD45RA resident leukocyte-derived lineages, mostly macrophages, in the early human pancreatic mesenchyme. These cells may have migrated in the tissue through the vascular system, attracted by constitutively expressed chemokines, and locally surviving through CSF1 signaling. The role of macrophages in epithelium/mesenchyme interaction-mediated pancreatic development remains to be demonstrated.

Biochemical investigations in rodents have shown that numerous mineralized matrix proteins share expression in bone, dentin, and cementum. Little information is available regarding the expression pattern of these proteins in human tissues, particularly during tooth formation. The aim of this study was to identify the expression pattern of the two major noncollagenous proteins of bone and dentin, osteocalcin (OC) and osteonectin (ON), in comparison to the dentin-specific protein, dentin sialophosphoprotein (DSPP). Mandibles from fetuses (5-26 weeks), neonate autopsies, forming teeth from 10-12-year-old patients, third molars extracted for orthodontic reasons, and bone tumors were collected with approval from the National Ethics Committee. Human OC, ON, and DSPP mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR) in fetal mandibles (5-11 weeks) and in primary cell cultures of dental pulp. In addition, OC, ON, and DSPP proteins were localized in forming human mineralized tissues using immunohistochemistry. In vivo, DSPP expression was associated with tooth terminal epithelial-mesenchymal interaction events, amelogenesis and dentinogenesis. Transient DSPP expression was seen in the presecretory ameloblasts with continuous expression in the odontoblasts. In contrast, both osteoblasts and odontoblasts showed a temporal gap between OC and ON expression in early development. ON was expressed in the initial stages of cytodifferentiation, whereas OC was expressed only during the later stages, especially in the teeth. At the maturation stage of enamel formation, both proteins were detected in odontoblasts and their processes within the extracellular matrix. In contrast to bone, OC was not localized extracellularly within the collagen-rich dentin matrix (predentin or intertubular dentin), but was found in the mature enamel. ON was present mostly in the nonmineralized predentin. These results demonstrate for the first time that both OC and ON are produced by human odontoblasts and determine the expression pattern of DSPP in human teeth, and suggest that OC and ON move inside the canalicule via odontoblast cell processes becoming localized to specific extracellular compartments during dentin and enamel formation. These distinct extracellular patterns may be related to the nature of DSPP, OC, and ON interactions with other matrix-specific macromolecules (i.e., amelogenin, dentin matrix protein-1) and/or to the polarized organization of odontoblast secretion as compared with osteoblasts.

Cytokines play a role in alopecia areata. We used immunohistochemical and in situ hybridization studies to demonstrate the persistence of pro-inflammatory as well as apoptotic mechanisms in skin biopsies from patients with chronic alopecia areata. In situ hybridization allows the visualization of the distribution of immunocompetent cells in vivo. We studied skin biopsies from 11 untreated alopecia areata patients and two normal controls. In situ hybridization was performed on frozen sections using 35S-radio-labeled riboprobes, specific for IL-1beta, IL-2, IL-6, INFgamma, and granzyme B mRNA. Immunohistochemistry was carried out using an anti-IL-1beta monoclonal antibody, and a monoclonal antibody directed against the human Fas protein. We demonstrated the presence of cells labeled with IL-1beta, IL-6, INFgamma, and granzyme B antisense probes. Similarly, cells labeled with anti-IL-1beta were found in 10 of 11 cases. The labeled cells were located in the mononuclear peri- and intrafollicular infiltrate. Cells expressing granzyme B were found in close contact with the follicle. Fas positivity was demonstrated in four of four cases at the level of the cytoplasmic membrane of the hair follicle keratinocytes. These results, based on visualizing the labeled cells, demonstrate that pro-inflammatory cytokines are produced by the mononuclear cell infiltrate in close contact with follicles in alopecia areata. Furthermore, they demonstrate for the first time that apoptotic mechanisms involving granzyme B and Fas-Fas ligand pathways may play a major role in the persistence of chronic alopecia areata.

Dominant megacolon (Dom) is one of four mutations in the mouse that can produce a phenotype similar to Hirschsprung disease in human. The Dom gene product is not known, and no candidate region has been defined for a possible human homolog. In this publication we report mapping the Dom locus with high definition, using several intra- and interspecific crosses and a set of 16 Chr 15-specific microsatellites flanking this locus.

The number, distribution, and phenotype of mononuclear cells infiltrating the allograft lung transplant were determined immunohistochemically with monoclonal antibodies directed against cellular antigens (CD3, CD4, CD8, CD22, CD25, CD16, CD56, CD68, HLA-DR) on frozen sections of transbronchial biopsies. Seventy-two transbronchial biopsies from 21 patients undergoing lung or heart-lung transplantation were evaluated histologically and immunohistologically in a prospective study. Four major results were obtained in the graft lung parenchyma: (1) whatever the histological grading of rejection, T lymphocytes expressing CD3 were present and in a significantly higher number than in control subjects (P < 0.0005); (2) there was a positive correlation between histological rejection and the number of CD3+, CD8+, CD25+, CD16+ cells (P < 0.01); (3) the CD4/CD8 ratio was inverted (0.52 +/- 0.04), with no correlation with the histological rejection; and (4) the number and location of CD3+, CD25+ cells did not correlate with CMV identification in bronchoalveolar lavage. Immunohistochemical criteria could be used for diagnosis of rejection in the management of heart-lung transplantation.

With a view to investigating the implication of IGF-binding protein-6 (IGFBP-6) in the growth of neuroblastomas, nude mice were injected with IGFBP-6-expressing or control IGR-N-91 human neuroblastoma cells and the resulting xenografts examined. Expression of IGFBP-3, IGFBP-4 and type 1 and type 2 IGF receptor messengers was similar in control tumours and equal-sized IGFBP-6-expressing tumours that had developed. IGF-II was more strongly expressed in control tumours, and IGFBP-6-expressing tumours contained less IGFBP-2 than controls. In both populations, there was a significant positive correlation between IGF-II and IGFBP-2 expression. In small IGFBP-6-expressing xenografts where tumour development had apparently been arrested, haematoxylin--eosin and TUNEL staining revealed numerous apoptotic cells. In situ hybridization indicated homogeneous distribution of the IGFBP-6 signal in test tumours. In cell culture, IGFBP-6-expressing cells expressed similar amounts of IGFBP-2, IGF-II and N-myc mRNAs as control cells; but media conditioned by IGFBP-6-expressing cells contained less intact IGFBP-2 protein, with no increase in its proteolytic fragment. In media treated with plasminogen, in which IGFBP-2 was proteolysed, IGFBP-6 was increased. With its especially strong affinity for IGF-II and its resistance to proteolysis, IGFBP-6 would act by sequestering IGF-II, hence inhibiting its mitogenic and anti-apoptotic effects. In excess, IGFBP-6 would displace IGF-II from IGFBP-2 whose potentiation of IGF-II action would cease and whose susceptibility to degradation would be increased. This study therefore shows that IGFBP-6 plays a role in neuroblastoma cell growth in vivo and in vitro and that stable overexpression of IGFBP-6 leads to alteration of the initial balance between the IGFBPs.

Post-transplant lymphoproliferative disorder (PTLD) after haemopoietic stem cell transplantation is a serious complication that occurs in 8-22% of patients with high-risk factors. We retrospectively investigated tolerance and efficacy of humanized anti-CD20 monoclonal antibody (rituximab) as first-line treatment in 12 children with B-cell PTLD. At diagnosis, eight patients had tumoral involvement. The other four patients had fever, associated with raised Epstein-Barr virus (EBV) viral load and monoclonal gammopathy. Rituximab was given at the dose of 375 mg/m2 once a week by intravenous infusion (1-9 infusions). Only 1/48 infusions was associated with a grade 2 clinical adverse event. Eight out of 12 (66%) patients responded to the treatment and were in complete remission. All patients without tumoral involvement responded to the treatment. A rapid decrease in fever within 1 week was observed in all responders. Non-responders did not show any clinical response during the first week. Tumoral involvement and immunodepression seemed to be more marked in non-responders. Rituximab was an effective and well-tolerated treatment of B-cell PTLD. Early treatment before tumoral involvement seemed to be the most effective approach. Lack of rapid response should lead to intensification of PTLD treatment. Pre-emptive treatment should be considered and evaluated in further longitudinal multicentre studies.

Cytochromes P4503A (the predominant CYP450 subfamily) and P-glycoprotein (P-gp, an efflux plasma membrane protein) are mainly located in enterocytes and hepatocytes and reduce the absorption of many drugs. During the neonatal period, a shift between CYP3A7, the fetal form, and CYP3A4 occurs in the liver but data on expression of the CYP3A/P-gp complex in the intestine are very limited. Localisation and expression of CYP3A and P-gp were studied in 59 normal duodenal biopsies from Caucasian children aged 1 month to 18 years. The immunoblot analysis (polyclonal antibody Nuage) showed that CYP3A was expressed in half the enterocytes until 6 months and in all the older children. P-gp (monoclonal antibody “C494”) was expressed at the apical surface of all enterocytes. mRNA quantification of the 3 CYP3A isoforms (3A4, 3A5, 3A7) and P-gp was performed using highly specific real time RT-PCR (normalisation with villin). CYP3A4 and CYP3A5 mRNA expression were high at birth and decreased afterwards (0.04 - 0.25 and 0.001 to 0.46 respectively, including expression of mutated allele CYP3A5*3), CYP3A7 mRNA was detected in 35% of the samples at a low level. P-gp mRNA was found variably expressed from 0.08 to 1 with a significant increase between 6 and 12 months of age. Our results showed that neonates and infants have a significant intestinal expression of CYP3A and P-gp mRNA. Additional studies are required to quantify protein activity and determine the clinical impact of these results. Clinical Pharmacology & Therapeutics (2004) 75, P50–P50; doi: 10.1016/j.clpt.2003.11.189

P-glycoprotein (P-gp) is an energy-dependent efflux pump expressed in the syncythiotrophoblast of human placenta throughout pregnancy, suggesting a potential functional importance of Pgp during pregnancy. Human data are limited and the effects of maternal treatment with corticosteroids in case of preterm labor have not known. After maternal consent, placenta from term pregnancies (n=20) or term pregnancies treated with betamethasone for preterm labor (n=43) were studied. Betamethasone (12 mg twice at 24 hour interval) was administered between 22 and 34 week's gestation, interval between end of treatment and birth was 35 ± 26 days. Immunohistochemical localisation by a sandwich staining method with Dako 3522 and F4 Kamiya monoclonal antibodies in paraffin embedded human placenta showned that P-gp is located in the endothelial cells without evidence for expression in the syncythiotrophoblast. P-gp mRNA and the constititively expressed mRNA 18S) were measured by RT-PCR and results expressed as the ratio of Pgp over 18S mRNA. PgP was highly expressed in the placenta but Pgp/18S mRNA ratio was not different at term between normal pregnancies and pregnancies treated with betamethasone. A acute effect of betamethasone on P-gp expression cannot be rulled out, as the free interval between betamethasone and birth was more than three weeks and will be investigated by additional studies. Clinical Pharmacology & Therapeutics (2004) 75, P73–P73; doi: 10.1016/j.clpt.2003.11.277