Zaki Kraiem

Impaired Light Chain Allelic Exclusion and Lack of Positive Selection in Immature B Cells Expressing Incompetent Receptor Deficient of CD19

Positive signaling is now thought to be important for B cell maturation, although the nature of such signals has not yet been defined. We are studying the regulatory role of B cell Ag receptor (BCR) signaling in mediating positive selection of immature B cells. To do so, we use Ig transgenic mice (3-83Tg) that are deficient in CD19, thus generating a monoclonal immature B cell population expressing signaling-incompetent BCR. Immature 3-83Tg CD19−/− B cells undergo developmental arrest in the bone marrow, allowing maturation only to cells that effectively compensate for the compromised receptor by elevated levels of BCR. We find that developmentally arrested 3-83Tg CD19−/− B cells fail to impose L chain allelic exclusion and undergo intensive V(D)J recombination to edit their BCR. Furthermore, immature 3-83Tg CD19−/− B cells, which were grown in vitro, failed to undergo positive selection and to survive when adoptively transferred into normal recipients. However, elevation of BCR expression levels, obtained by transgene homozygosity, effectively compensated for the compromised BCR and completely restored BCR-mediated Ca2+ influx, allelic exclusion, and positive selection. Our results suggest that the BCR signaling threshold mediates positive selection of developing B cells, and that a receptor-editing mechanism has an important role in rescuing cells that fail positive selection because of incompetent receptors.

THYROTROPHIN RECEPTOR BLOCKING ANTIBODIES: INCIDENCE, CHARACTERIZATION AND IN‐VITRO SYNTHESIS

The prevalence and characteristics of TSH receptor blocking activity were examined in patients with different thyroid disorders. Studies were also performed on the in‐vitro synthesis and immunoregulation of the blocking antibody. Blocking activity was tested by measuring the inhibition of TSH‐stimulated cAMP production of cultured human thyroid cells by patient immunoglobulin (Ig) preparations. The following patients were investigated (number of cases in parentheses): Hashimotos thyroiditis (33); primary myxoedema (17); euthyroid ophthalmopathy (8); active Graves’disease (19); cold nodule (5); non‐toxic goitre (14); toxic adenoma (8); toxic multinodular goitre (9) and 22 normal controls. TSH receptor blocking activity was only detected in primary hypothyroidism with the following characteristics: (i) Such activity was present in only 16% of the patients (both goitrous, i.e. Hashimotos thyroiditis, and non‐goitrous, i.e. primary myxoedema), and in three patients with previously active Graves’hyperthyroidism who had become hypothyroid. (ii) Blocking activity seems to be associated with the Ig fraction of serum as indicated by protein A adsorption, (iii) The block‐positive samples did not bind l25I‐TSH, which seems to rule out an antibody directed against TSH. (iv) The specificity of the blocking activity seems to be directed towards the TSH‐(thyroid stimulating immunoglobulin, TSI) receptor‐mediated cAMP response since no inhibition of prostaglandin E1‐stimulated cAMP production was found. Moreover, all cases in which an inhibitory effect was demonstrated towards TSH also exhibited blocking of TSI‐stimulated cAMP, with a high correlation between the degree of inhibition of the TSH to that of the TSI response (r= 0.89, P<0.001, n= 11). The blocking activity may contribute to the pathogenesis of some cases of primary hypothyroid autoimmune thyroiditis, both goitrous and non‐goitrous, as well as in the evolution of hyper‐ to hypothyroidism. By culturing peripheral blood lymphocytes, as well as B/T lymphocyte co‐cultures isolated from three patients with blocking activity present in serum, the in‐vitro synthesis of the blocking antibody was demonstrated for the first time. Moreover, in‐vitro secretion of the antibody by patients B lymphocytes, as well as T cell regulation of autoantibody production, were also shown.

Decreased expression of tissue inhibitor of matrix metalloproteinases in follicular fluid from women with polycystic ovaries compared with normally ovulating patients undergoing in vitro fertilization

OBJECTIVE To compare activity of matrix metalloproteinases (MMP) and expression of their tissue-specific inhibitor (TIMP) in the follicular fluid of normally ovulating women and women with the polycystic ovary syndrome (PCOS). DESIGN Prospective study. SETTING IVF unit and endocrine research unit. PATIENT(S) Fourteen patients undergoing IVF treatment (seven with normal ovulation and seven with PCOS). MAIN OUTCOME MEASURE(S) Activity of MMP-2 and MMP-9 and expression of MMP-1, TIMP-1, and TIMP-2 was measured in follicular fluid of the leading follicles by using gel zymography and immunoblot analysis. RESULT(S) The activity of MMP-2 and MMP-9 and expression of MMP-1 was similar in follicular fluid of normally ovulating patients and patients with PCOS. Significantly lower expression of TIMP-1 was found in follicular fluid of patients with PCOS women compared with normally ovulating patients. CONCLUSION(S) Because MMPs and TIMPs play a role in the physical and chemical structure of the follicular compartment, the decreased expression of TIMP in patients with PCOS may be part of a compensatory process to overcome the physical properties of the thick ovarian capsule.

Stimulation of Fos- and Jun-related Genes During Distraction Osteogenesis

Bone cells respond to mechanical stimulation by gene expression. The molecular events involved in the translation of mechanical stimulation into cell proliferation and bone formation are not yet well understood. We looked for the expression of early-response genes of the AP-1 transcription factor complex in an in vivo bone regeneration system subjected to mechanical forces because these genes were found to be related to mechanotransduction and important for bone development. Sheep maxillary bone was distracted daily for 15 days. c-Jun and c-Fos were evaluated by Northern blotting analysis and immunohistochemistry in biopsy specimens removed at 8 and 15 days and were compared with post-osteotomy but not distracted repair tissue. Elevated levels of c-Jun and c-Fos mRNA were found after 8 days of distraction. Likewise, mesenchyme-like and fibroblast-like cells composing the 8-day distracted regeneration tissue showed increases in the intensity of immunostaining compared to cells in the corresponding non-distracted fracture repair tissue. After 15 days of distraction, when bone trabeculae start to form distally and proximally in the distracted regeneration tissue, mostly preosteoblasts and osteoblasts retained c-Fos and c-Jun immunoreactivity, similar to bone-associated cells in control non-distracted fracture repair tissue. We propose that the elevated expression of c-Jun and c-Fos is related to mechanical stimulation in this in vivo bone regeneration system.

Anti-thyroid cancer properties of a novel isoflavone derivative, 7-(O)-carboxymethyl daidzein conjugated to N-t-Boc-hexylenediamine in vitro and in vivo

The incidence of thyroid cancer is up to 3 folds higher in women than in men, suggesting that estrogenic effects may be involved in the pathogenesis of this malignancy. Here, we explore whether or not human thyroid cancer cell growth can be curbed by a novel isoflavone derivative generated in our laboratory, the N-t-Boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc). With the exception of the follicular cancer cell line WRO, estrogen receptor (ER)α mRNA was only marginally expressed in cell lines derived from papillary (NPA), follicular (MRO), anaplastic thyroid carcinoma (ARO) such that the expression of estrogen receptor (ER) βmRNA was more abundant than that of ERα mRNA in these cell types. Estradiol-17β (E2; 0.03-300nmol/l) per se increased proliferation in all four cell-types. The ERβ-specific agonist DPN increased [(3)H]-thymidine incorporation in all four thyroid cancer cell lines, whereas the ERα-specific agonist PPT increased growth only in NPA and WRO. By contrast, cD-tboc, derived from the weak estrogen daidzein, did not cause cell growth and dose-dependently diminished cell growth in all four cell lines via apoptosis and not necrosis, as detected by the release of histone-DNA fragments. The cytotoxic growth inhibitory effect of cD-tboc in these cells was modulated by E2 and the general caspase inhibitor Z-VAD-FMK, and the magnitude of this salvage was cell type-and dose-dependent. When nude mice carrying ARO thyroid xenografts were treated with cD-tboc, tumor volume decreased significantly, and no apparent toxicity was observed. These results suggest that cD-tboc may be a promising agent for therapy of thyroid carcinoma either alone or in combination with existing cytotoxic drugs.

Growth inhibition of human thyroid carcinoma and goiter cells in vitro by the isoflavone derivative 7-(O)-carboxymethyl daidzein conjugated to N-t-boc-hexylenediamine.

BACKGROUND Estrogens may enhance thyroid cancer cell growth. We have recently reported that a novel isoflavone-derived anti-estrogenic compound developed in our laboratory, the N-t-boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc), can induce apoptosis and retard growth in human thyroid carcinoma cell lines through inhibitory interaction on estrogen receptor β. Here we tested the hypothesis that cD-tboc can likewise retard cell growth in cultured human thyroid papillary carcinoma cells, normal thyroid cells, and goiter cells removed during thyroidectomy. METHODS In vitro experiments in cultured human thyroid normal, goiter, and papillary thyroid carcinoma (PTC) cells were performed. Estrogen receptors α and β (ERα and ERβ), DNA synthesis and creatine kinase (a marker of estrogenic genomic response), and the effects of cD-tboc on DNA synthesis in cultured human PTC cells were assessed. RESULTS First, all cell types thus harvested and grown in culture expressed both ERα and ERβ, with a variably higher abundance of ERβ over ERα seen in the goiter and PTC cells, but not in the normal thyroid cells. Second, DNA synthesis and creatine kinase were increased in response to estradiol-17β (E2), the ERα agonist propyl-pyrazole-trisphenol as well as the ERβ agonist diarylpropionitrile. Third, cD-tboc dose-dependently inhibited DNA synthesis in cultured human PTC cells (-65%) and to a lesser extent in goiter cells (∼-30%). CONCLUSION This study provides the first evidence that cD-tboc can act to inhibit growth in primary cultures of human PTC cells and goiter cells removed during thyroidectomy. Whether this can be utilized for the treatment of human thyroid cancer and/or goiter remains to be explored.

Urinary and blood levels of adenosine 3',5'-monophosphate, phosphorus and calcium in infants.

The changes during the first year of life in urinary and nephrogenous cAMP levels were determined in 152 normal, full-term infants using spot urine samples. The use of spot tests simplifies considerably the logistics of the cAMP determination which can be used for the investigation of parathyroid function. Levels of urinary and nephrogenous cAMP (expressed both as a function of creatinine excretion as well as glomerular filtration rate) rose from birth to 1 year of age, whereas plasma cAMP levels did not change significantly. Levels of cAMP (both urinary and nephrogenous) were significantly correlated with age, length, weight, and urinary phosphorus concentrations of the infants. No significant correlation could be observed, however, between serum phosphorus or serum and urinary calcium on the one hand, and urinary or nephrogenous cAMP on the other.

The influence of cryopreservation on the ultrastructural morphology of human thyroid cells

The purpose of this study was to investigate the effects of the freeze-thaw procedure on the ultrastructural features of human thyroid cells. Four different stages of thyroid cell preparation were compared: (1) fresh surgical tissue, serving as control, (2) cell suspension before freezing, (3) cell suspension after thawing, and (4) monolayer cell culture, obtained from cells after thawing. Electron microscopic examination of cells from each stage showed that the freeze-thaw procedure used caused only minor intracellular alterations restricted to mitochondria. Some of these organelles showed low-amplitude swelling or occasionally appeared condensed. These ultrastructural changes were not paralleled by a decrease in the vitality or sensitivity of the cryopreserved cells to stimulating agents.

Cellular immune functions in patients with primary hyperparathyroidism: Effects of histamine and cimetidine

The possibility of a link between parathyroid hyperfunction and cellular immune functions was studied in primary hyperparathyroid (1 degree HP) patients. The effect of cimetidine on lymphocyte functions in 1 degree HP patients and control subjects was also investigated. Histamine-induced suppressor activity of lymphocytes from 1 degree HP patients was significantly greater than that of controls. Cimetidine addition to both normal and 1 degree HP lymphocyte cultures abolished histamine-induced suppression. In vivo administration of cimetidine, while ineffective towards normal lymphocytes, depressed phytohemagglutinin stimulation of 1 degree HP lymphocytes, indicating possible immunological damage caused by this drug, which is frequently used in the treatment of patients with 1 degree HP.