Impact of incidental parathyroidectomy and mediastinal-recurrent cellular and lymph-node dissection on parathyroid function after total thyroidectomy.

OBJECTIVES: To determine the impact of incidental parathyroidectomy and mediastinal-recurrent cellular and lymph-node dissection on parathyroid function after total thyroidectomy. MATERIAL AND METHODS: A single-center retrospective study was conducted for a 5-year period in a university hospital center, including 605 patients undergoing total thyroidectomy, 52 of whom had mediastinal-recurrent cellular and lymph-node dissection. ENDPOINTS: The main endpoint was intraoperative number of parathyroid glands as predictor of parathyroid hormone (PTH) level and postoperative hypocalcemia. The secondary endpoint was the correlation between associated mediastinal-recurrent cellular and lymph-node dissection and incidental parathyroidectomy and its impact on PTH level and calcemia in the immediate postoperative period and at 1 month. RESULTS: 161 patients (26.61%) showed hypocalcemia in the immediate postoperative period and 12 (1.98%) at 1 month. Mediastinal-recurrent cellular and lymph-node dissection increased incidental parathyroidectomy risk 4.6-fold. Mediastinal-recurrent cellular and lymph-node dissection was associated with a statistically "suggestive" decrease in day-1 calcemia (P=0.03), and no significant decrease at 1 month (P=0.52). Incidental parathyroidectomy (6.7% of cases with parathyroidectomy versus 1.3% without) did not significantly increase the rate of early hypocalcemia (P=0.28), but was associated with a "suggestive" worsening at 1 month (P=0.02). CONCLUSION: Hypocalcemia after total thyroidectomy is a complex, probably multifactorial issue. Systematic parathyroid gland identification is not recommended due to the increased risk of gland lesion, mainly by devascularization. Incidental parathyroidectomy may induce hypocalcemia at 1 month postoperatively (statistically "suggestive" association).

Head and neck squamous cell carcinoma and metachronous second primaries.

OBJECTIVES: To assess the rate of second (or more) primaries after treatment for head and neck squamous cell carcinoma (HNSCC), and survival compared to patients with a single head and neck cancer. MATERIAL AND METHOD: A single-center retrospective study was performed in a University Hospital Center in 541 patients between 2002 and 2010. RESULTS: One hundred and forty-one patients (26.06%) presented 172 metachronous cancers. Overall 5-year survival was 20.3% with and 38.1% without metachronous cancer. Median and mean survival were respectively 21.9 and 51 months in patients with a single cancer, versus 13.9 and 26.5 months in case of metachronous cancer. Specific survival was comparable to overall survival. All-cause and specific survival were significantly poorer in metachronous cancer (P=0.001; log-rank α=0.05). CONCLUSION: At least a quarter of HNSCC patients go on to develop a metachronous second primary. These are of poor prognosis, whatever their location.

Ectopic retroviral expression of LMO2, but not IL2Rgamma, blocks human T-cell development from CD34+ cells: implications for leukemogenesis in gene therapy.

The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rgamma), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rgamma had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rgamma may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth.

Wheat germ agglutinin affinity of murine hemopoietic stem cell subpopulations is an inverse function of their long-term repopulating ability in vitro and in vivo.

Hemopoietic stem cells show extensive heterogeneity with respect to their proliferative potential and activity. We have recently reported that the accepted technique for sorting stem cells on the basis of high affinity for the lectin wheat germ agglutinin (WGA) did not select for cells initiating long-term production of new stem cells on a stromal layer in vitro. We have therefore reinvestigated the expression of cell surface sialic acid residues in the hemopoietic stem cell compartment by sorting murine bone marrow cells on the basis of affinity for WGA. Frequency analysis of long-term bone marrow culture initiating stem cells was done using the cobblestone-area-forming cell (CAFC) assay with limiting dilution set-up. In vivo stem cell quality was determined by spleen colony formation, marrow-repopulating ability (MRA) and long-term repopulating ability (LTRA) using sex-mismatched hemopoietic chimerism. The data indicate that MRA and LTRA in vivo and in vitro are among the most WGA-dim cells. In contrast, the enrichment factors for splenic colony-forming units (CFU-S) at day 12 and transient CAFC increase with increasing WGA affinity. These characteristics allowed us to concentrate LTRA cells 590- to 850-fold over their activity in normal bone marrow without significant enrichment of day-12 CFU-S. The data reveal that WGA affinity is an inverse function of the primitiveness of murine hemopoietic stem cells and that long-term production of blood cells in vivo and in vitro is provided for by primitive cells that are physically separable from the vast majority of day-12 CFU-S. In addition the data reveal, that the CAFC frequency at day 28-35 of a graft strongly correlates with the number of cells required to induce 40% donor-type chimerism at 15 months post-transplantation and thus predicts the in vivo LTRA of a graft.

Loss of long-term repopulating ability in long-term bone marrow culture.

We have studied the maintenance of stem cells with long-term multilineage repopulating ability from murine bone marrow, cultured on a pre-established bone marrow-derived stromal cell layer, both in a qualitative and quantitative way. Female bone marrow cells were cultured for a period of 1-4 weeks and compared with uncultured cells for their ability to establish and maintain a level of 50% chimerism in a sex-mismatched bone marrow transplantation model. Chimerism was determined in nucleated cells using fluorescence in situ hybridization with a murine Y-chromosome-specific probe. We observed a rapid decline in the ability of cultured marrow cells to repopulate the blood, bone marrow, spleen, and thymus of sublethally irradiated male recipients. After 4 weeks of culture only 5% of the long-term repopulating ability of the inoculated bone marrow cells remained. The remaining long-term repopulating cells, however, had similar qualities to establish and maintain long-term engraftment compared to fresh bone marrow, as judged from their ability to give stable chimerism over a period of 6 months. These observations are relevant for the therapeutic applications of long-term bone marrow cultures in purging protocols prior to autologous bone marrow transplantation of acute and chronic myeloid leukemic patients, and for the use of long-term marrow cultures when introducing foreign genetic material in hematopoietic stem cells.

DNA microarrays for comparison of gene expression profiles between diagnosis and relapse in precursor-B acute lymphoblastic leukemia: choice of technique and purification influence the identification of potential diagnostic markers.

Microarrays for gene expression profiling are rapidly becoming important research tools for the identification of novel markers, for example, for novel classification of leukemias and lymphomas. Here, we review the considerations and infrastructure for microarray experiments. These considerations are illustrated via a microarray-based comparison of gene expression profiles of paired diagnosis-relapse samples from patients with precursor-B acute lymphoblastic leukemia (ALL), who relapsed during therapy or after completion of treatment. Initial experiments showed that several seemingly differentially expressed genes were actually derived from contaminating non-leukemic cells, particularly myeloid cells and T-lymphocytes. Therefore, we purified the ALL cells of the diagnosis and relapse samples if their frequency was lower than 95%. Furthermore, we observed in earlier studies that extra RNA amplification leads to skewing of particular gene transcripts. Sufficient (non-amplified) RNA of purified and paired diagnosis-relapse samples was obtained from only seven cases. The gene expression profiles were evaluated with Affymetrix U95A chips containing 12 600 human genes. These diagnosis-relapse comparisons revealed only a small number of genes (n=6) that differed significantly in expression: mostly signaling molecules and transcription factors involved in cell proliferation and cell survival were highly upregulated at relapse, but we did not observe any increase in drug-resistance markers. This finding fits with the observation that tumors with a high proliferation index have a poor prognosis. The genes that changed between diagnosis and relapse are currently not in use as diagnostic or disease progression markers, but represent potential new markers for such applications. Leukemia (2003) 17, 1324-1332. doi:10.1038/sj.leu.2402974

Differential adherence of murine hematopoietic stem cell subsets to fibronectin.

Growth and differentiation of hematopoietic stem cells occur in close contact with the cells and extracellular-matrix (ECM) proteins of the hematopoietic microenvironment. We observed the role of fibronectin, a matrix glycoprotein proposed to be involved in the attachment of hematopoietic cells and in the binding of stem cell subsets to bone marrow stroma, for this study. Murine bone marrow cells (BMC) were allowed to adhere to surfaces coated with human plasma fibronectin. Using the cobblestone-area-forming cells (CAFC) assay, adherent and nonadherent cell fractions were tested for their quantity of primitive and less primitive stem cell subsets. The CAFC assay is based on a time-dependent clone formation in pre-established, bone marrow-derived, irradiated stromal layers under limiting dilution conditions, and it allows in vitro enumeration of day-12 colony-forming unit-spleen (CFU-S12) (CAFC-10) and more primitive cells with long-term repopulating abilities (LTRA) (CAFC-28/35). We observed that the majority of primitive CAFC-28/35 adhered to fibronectin, while only a minority of CFU-S-like CAFC-10 did. The adherence of primitive stem cells to fibronectin could partially be blocked by high molar concentrations of oligopeptides containing the essential amino acid sequence of the central cell-binding domain of fibronectin, RGD. Adherence of the small subpopulation of CAFC-10 to fibronectin could almost entirely be prevented by oligopeptides organized in a specific fashion. These data suggest a role for RGD-binding integrins in the adherence of hematopoietic stem cells.

Selective advantage of normal erythrocyte production after bone marrow transplantation of alpha-thalassemic mice.

Anemia resulting from alpha-thalassemia in mice was corrected by transplantation of normal bone marrow cells following sublethal total body irradiation, resulting in partial hematopoietic chimerism with a preponderance of normal peripheral blood red cells. Peripheral blood red cell chimerism in recipients of graded numbers of bone marrow cells from sex-mismatched donors, determined by cytometric analysis, was directly compared with immature hematopoietic cell (CFU-S) chimerism and peripheral blood white cell chimerism. The latter two were assessed by fluorescent in situ hybridization with a murine Y-chromosome-specific probe. Peripheral blood white cell chimerism consistently corresponded with immature hematopoietic cell chimerism, emphasizing the selective advantage of normal red cell production in partially chimeric alpha-thalassemic mice.

Differential ultraviolet-B-induced immunomodulation in XPA, XPC, and CSB DNA repair-deficient mice.

Ultraviolet B irradiation has serious consequences for cellular immunity and can suppress the rejection of skin tumors and the resistance to infectious diseases. DNA damage plays a crucial role in these immunomodulatory effects of ultraviolet B, as impaired repair of ultraviolet-B-induced DNA damage has been shown to cause suppression of cellular immunity. Ultraviolet-B-induced DNA damage is repaired by the nucleotide excision repair mechanism very efficiently. Nucleotide excision repair comprises two subpathways: transcription-coupled and global genome repair. In this study the immunologic consequences of specific nucleotide excision repair defects in three mouse models, XPA, XPC, and CSB mutant mice, were investigated. XPA mice carry a total nucleotide excision repair defect, whereas XPC and CSB mice only lack global genome and transcription-coupled nucleotide excision repair, respectively. Our data demonstrate that cellular immune parameters in XPA, XPC, and CSB mice are normal compared with their wild-type (control) littermates. This may indicate that the reported altered cellular responses in xeroderma pigmentosum patients are not constitutive but could be due to external factors, such as ultraviolet B. Upon exposure to ultraviolet B, only XPA mice are very sensitive to ultraviolet-B-induced inhibition of Th1-mediated contact hypersensitivity responses and interferon-gamma production in skin draining lymph nodes. Lipopolysaccharide-stimulated tumor necrosis factor alpha and interleukin-10 production are significantly augmented in both XPA and CSB mice after ultraviolet B exposure. Lymph node cell numbers were increased very significantly in XPA, mildly increased in CSB, and not in XPC mice. In general XPC mice do not exhibit any indication of enhanced ultraviolet B susceptibility with regard to the immune parameters analyzed. These data suggest that both global genome repair and transcription-coupled repair are needed to prevent immunomodulation by ultraviolet B, whereas transcription-coupled repair is the major DNA repair subpathway of nucleotide excision repair that prevents the acute ultraviolet-B-induced effects such as erythema.

Differential mRNA expression and production of interleukin-4 and interferon-gamma in stimulated peripheral blood mononuclear cells of house-dust mite-allergic patients.

Optimal culture conditions were established for the analysis of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression and protein production, as well as proliferative capacity of peripheral blood mononuclear cells (PBMC). These culture conditions permitted the analysis of differences in the responses of house-dust mite (HDM) allergic patients and healthy controls after polyclonal and allergen-specific stimulation. Proliferative responses were optimal when PBMC were cultured in RPMI, whereas for studying mRNA expression by RT-PCR and protein production by ELISA, PBMC should be stimulated in Yssels's medium. Blood holding period influenced the cytokine mRNA expression and proliferative capacity of primarily the unstimulated cells. It is thus crucial to isolate PBMC as soon as possible, and in any event no later than 7 hours after blood collection. Proliferative responses to Dermatophagoides pteronyssinus-extract were observed in HDM allergic patients (mean stimulation index (SI) = 5.3+/-0.75), but not in non-allergic subjects (mean SI = 2.3+/-0.21). After D. pteronyssinus-specific stimulation, IL-4 mRNA expression was significantly (p = 0.03) increased in HDM-allergic subjects compared to non-allergic subjects. No significant differences were found in IFN-gamma mRNA expression between HDM-allergic and non-allergic subjects. Both IFN-gamma (p = 0.04) and IL-4 (p = 0.06) protein production were increased after D. pteronyssinus-specific stimulation in HDM-allergic subjects compared to non-allergic subjects. Our data suggest activation of both Th1 and Th2-like cells, as well as CD8+ T cells in allergic patients. Furthermore, analysis of possible functional differences in PBMC between allergic and non-allergic patients, necessitates polyclonal and allergen-specific stimulation of PBMC. Moreover, proliferative responses as well as cytokine mRNA expression and protein production should be studied under optimal culture conditions to highlight the often subtle differences.

Correction of murine Rag1 deficiency by self-inactivating lentiviral vector-mediated gene transfer.

Severe combined immunodeficiency (SCID) patients with an inactivating mutation in recombination activation gene 1 (RAG1) lack B and T cells due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment option for RAG-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy has proven challenging. As a preclinical model for RAG-SCID, we used Rag1-/- mice and lentiviral self-inactivating (SIN) vectors harboring different internal elements to deliver native or codon-optimized human RAG1 sequences. Treatment resulted in the appearance of B and T cells in peripheral blood and developing B and T cells were detected in central lymphoid organs. Serum Ig levels and Ig and TCR Vß gene segment usage was comparable to wild-type (WT) controls, indicating that RAG-mediated rearrangement took place. Remarkably, relatively low frequencies of B cells produced WT levels of serum immunoglobulins. Upon stimulation of the TCR, corrected spleen cells proliferated and produced cytokines. In vivo challenge resulted in production of antigen-specific antibodies. No leukemia development as consequence of insertional mutagenesis was observed. The functional reconstitution of the B- as well as the T-cell compartment provides proof-of-principle for therapeutic RAG1 gene transfer in Rag1-/- mice using lentiviral SIN vectors.

Correction of B-cell development in Btk-deficient mice using lentiviral vectors with codon-optimized human BTK.

X-linked agammaglobulinemia (XLA) is the most common primary immunodeficiency (PID) in man and caused by mutations in the Bruton's tyrosine kinase (BTK) gene. XLA is characterized by a B-cell differentiation arrest in bone marrow, absence of mature B cells and immunoglobulins (Igs), and recurrent bacterial infections. We used self-inactivating lentiviral vectors expressing codon-optimized human BTK under the control of three different ubiquitous or B cell-specific promoters. Btk-/- mice engrafted with transduced cells showed correction of both precursor B-cell and peripheral B-cell development. Lentiviral vectors containing the wildtype BTK sequence did not correct the phenotype. All treated mice with codon-optimized BTK exhibited the recovery of B1 cells in the peritoneal cavity, and of serum IgM and IgG3 levels. Calcium mobilization responses upon B-cell receptor stimulation as well as in vivo responses to T cell-independent antigens were restored. Viral promoters overexpressing BTK >100-fold above normal resulted in erythro-myeloid proliferations independent of insertional mutagenesis. However, transplantation into secondary Btk-/- recipients using cellular promoters resulted in functional restoration of peripheral B cells and IgM levels, without any adverse effects. In conclusion, transduction of human BTK corrects B-cell development and antigen-specific antibody responses in Btk-/- mice, thus indicating the feasibility of lentiviral gene therapy for XLA, provided that BTK expression does not vastly exceed normal levels.

Analysis of cyt0kine gene expression in stimulated T cells of small children by semi-quantitative PCR.

Only limited amounts of peripheral blood samples can be obtained from small children. Therefore, a polymerase chain reaction (PCR) aided analysis of cytokine gene expression by PBMC or T cells is a valuable tool. We present a combination of procedures to obtain an accurate estimation of the expression of the cytokines IL-4 and IFN-gamma. This can be performed on T cells purified from blood samples of up to 5 ml in volume from children aged 0-4 years with allergic asthma and atopic dermatitis. This procedure includes multiple sampling of PCR products to determine the linear phase of the PCR; inter-experiment correction using a helper T-cell clone, expressing both IL-4 and IFN-gamma; interpatient correction by comparing the expression of a housekeeping gene (HPRT); and finally the development of specific software to analyse densitometric data obtained by scanning photographs of agarose gels, separating PCR products. In this way it is possible to study cytokine gene expression from a very small amount of material.

Transferrin receptor expression and the regulation of placental iron uptake.

Placental transferrin receptors, located at the apical side of syncytiotrophoblast, mediate placental iron uptake. Regulation of transferrin receptors on the fetal-maternal exchange area could be a major determinant in the regulation of trans-placental iron transport. Transferrin receptor expression in cultured human term cytotrophoblasts is on a much lower level than in choriocarcinoma cells, with a higher proportion of receptors located on the cell surface. Differentiation of cells, either due to longer culture periods or to 8-bromo-cAMP treatment does not lead to an increase of transferrin receptor expression. In vitro, the level of expression is largely regulated by the cellular density in the culture dishes. Low cellular occupancy of the dish leads to a high level of transferrin receptors. Treatment with iron-sources results in a down regulation of transferrin receptors. Thus, though the level of transferrin receptors in cultured normal trophoblast is at a constant level, unaffected by differentiation, high levels of maternal transferrin-iron availability can lead to a decrease in placental iron uptake. This feed-back mechanism makes placental iron uptake independent of maternal iron stores.

T cell subsets and cytokines in allergic and non-allergic children. I. Analysis of IL-4, IFN-gamma and IL-13 mRNA expression and protein production.

Interleukin 4 (IL-4) and IL-13 are key cytokines inducing switching to immunoglobulin E (IgE), whereas interferon gamma (IFN-gamma) acts inhibitory on this process. We analysed whether differences existed in IL-4, IFN-gamma and IL-13 mRNA expression and protein production between T cells of children with allergic and non-allergic asthma, atopic dermatitis and health control children. IL-4 mRNA expression was increased in stimulated T cells of children with allergic asthma and atopic dermatitis, but not in those with non-allergic asthma as compared with healthy controls. Thus the increase in IL-4 expression can be considered as an underlying mechanism of the allergic disease process and not so much of the asthmatic state of the children. In unstimulated T cells of children with atopic dermatitis increased IFN-gamma mRNA expression with a reduced IFN-gamma protein production was found, indicating a post-translational defect in IFN-gamma. Differences in IL-13 expression between the groups were not significant, but IL-13 was significantly correlated with the height of the radio-allergo-sorbent test (RAST) class and with the severity scoring of atopic dermatitis (SCORAD) index. This indicates the clinical relevance of IL-13 for the degree of allergen-specific sensitization and severity of atopic dermatitis. In conclusion, the imbalance in IL-4 and IFN-gamma secretion in patients with atopic dermatitis may reflect general T cell activation in the presence of an intrinsic defect of IFN-gamma secretion.