18F-FDG PET/CT for the pre-surgical localization of epileptogenic focus among paediatric patients with drug resistant epilepsy in Malaysia: perspective of a nuclear medicine physician.

BACKGROUND: Scalp video electroencephalography monitoring (VEM) and brain MRI sometime fail to identify the epileptogenic focus (EF) in patients with drug resistant epilepsy (DRE). 18F-FDG PET/CT has been shown to improve the detection of EF in patients but is not widely used in Malaysia. Thus, the objective of this study was to identify whether 18F-FDG PET/CT conferred an added benefit in the pre-surgical evaluation of DRE. METHODS: Retrospective review of 119 consecutive paediatric patients referred for 18F-FDG-PET/CT at the Department of Nuclear Medicine of the National Cancer Institute, Putrajaya. All had DRE and underwent evaluation at the Paediatric Institute, Hospital Kuala Lumpur. Visually detected areas of 18F-FDG-PET/CT hypometabolism were correlated with clinical, MRI and VEM findings. RESULTS: Hypometabolism was detected in 102/119 (86%) 18FFDG- PET/CT scans. The pattern of hypometabolism in 73 patients with normal MRI was focal unilobar in 16/73 (22%), multilobar unilateral in 8/73 (11%), bilateral in 27/73 (37%) and global in 5/73 (7%) of patients; whilst 17/73 (23%) showed normal metabolism. In 46 patients with lesions on MRI, 18F-FDG-PET/CT showed concordant localisation and lateralization of the EF in 30/46 (65%) patients, and bilateral or widespread hypometabolism in the rest. Addition of 18FFDG PET/CT impacted decision making in 66/119 (55%) of patients; 24/73 with non-lesional and 30/46 patients with lesional epilepsies were recommended for surgery or further surgical work up, whilst surgery was not recommended in 11/46 patients with lesional epilepsy due to bilateral or widespread hypometabolism. 25 patients subsequently underwent epilepsy surgery, with 16/25 becoming seizure free following surgery. CONCLUSION: 18F-FDG-PET/CT has an added benefit for the localization and lateralization of EF, particularly in patients with normal or inconclusive MRI.

Features of post-radioiodine whole body scan in non-invasive Follicular Thyroid Neoplasm with papillary-like nuclear features (NIFTP).

Recently, encapsulated follicular variant of papillary thyroid carcinoma has been reclassified as non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) to emphasize the benign nature of this entity. In our institution, we have assessed 455 patients treated with radioiodine ablation for differentiated thyroid carcinoma and 20 of them were retrospectively found to fulfill the new NIFTP criteria. There was no evidence of metastasis on post radioiodine whole body scans for NIFTP cases and these patients were in remission subsequently. The benign features of these patients' whole body scans and good clinical outcome following treatment further support NIFTP as a low risk thyroid neoplasm.

Simvastatin inhibits the expression of inflammatory cytokines and cell adhesion molecules induced by LPS in human dental pulp cells.

AIM: To investigate the effect of simvastatin on lipopolysaccharide (LPS)-stimulated inflammatory cytokines, cell adhesion molecules and nuclear factor-κB (NF-κB) transcription factors in human dental pulp cells (HDPCs). METHODOLOGY: The effect of LPS and simvastatin on human dental pulp cell (HDPCs) viability was measured using a 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (MTT) assay. The expression of inflammatory cytokines and cell adhesion molecules was evaluated by reverse-transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. NF-κB transcription factors were evaluated by Western blot analysis. Statistical analysis was performed with analysis of variance (anova). RESULTS: The viability of cells exposed to different concentrations of E. coli LPS, P. gingivalis LPS and simvastatin was not significantly different compared with that of control cells (P > 0.05). LPS significantly increased interleukin (IL)-1ß (P < 0.05) and IL-6 mRNA expression (P < 0.05) and vascular cell adhesion molecule-1 (VCAM-1) (P < 0.05) and intercellular adhesion molecule-1 (ICAM-1) protein expression (P < 0.05) in HDPCs. Treatment with simvastatin significantly attenuated LPS-stimulated production of IL-1ß, IL-6, VCAM-1 and ICAM-1 (P < 0.05). Treatment with simvastatin decreased LPS-induced expression of p65 and phosphorylation of IκB and also significantly decreased the phosphorylation of p65 and IκB in the cytoplasm and the level of p65 in the nucleus (P < 0.05). CONCLUSIONS: Simvastatin has a suppressing effect on LPS-induced inflammatory cytokine, cell adhesion molecules and NF-κB transcription factors in HDPCs. Therefore, simvastatin might be a useful candidate as a pulp-capping agent in vital pulp therapy.

Transcriptional factor ATF6 is involved in odontoblastic differentiation.

ATF6 is an endoplasmic reticulum (ER) membrane-bound transcription factor that regulates various cellular functions. The purpose of this study was to investigate the role of ATF6 in odontoblast differentiation. Rat tooth germs were isolated, changes in gene expression were evaluated over time, and localization of ATF6 was determined by immunohistochemistry. Human dental pulp cells (HDPCs) were cultured with 50 µg/mL ascorbic acid and 5 mmol/L ß-glycerophosphate or 100 ng/mL bone morphogenetic protein 2 to induce differentiation. Translocation of ATF6 was observed by immunofluorescence and confocal microscopy. Overexpression of ATF6 was performed with an adenoviral vector. Matrix mineralization was evaluated by alizarin red staining. Immunoreactivity to anti-ATF6 was observed in the odontoblastic layer of the molar tooth germ, and expressions of ATF6, dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) increased gradually during tooth germ development. When HDPCs were cultured in differentiation media, ATF6, DSPP, and DMP1 expression increased with the expression of unfolded protein response (UPR) markers, BiP and CHOP. Immunofluorescence results showed that ATF6 protein moved from cytoplasm to nucleus when cells were exposed to differentiation media. Notably, overexpression of ATF6 increased DSPP and DMP1 expression, alkaline phosphatase (ALP) activity, and matrix mineralization in HDPC cultures. Inhibition of ATF6 decreased ALP activity and mineralization. These results suggest that ER membrane-bound transcriptional factor ATF6 may be involved in odontoblastic differentiation.

Imatinib mesylate (Gleevec) in advanced breast cancer-expressing C-Kit or PDGFR-beta: clinical activity and biological correlations.

BACKGROUND: Novel molecular therapies for metastatic breast cancer (MBC) are necessary to improve the dismal prognosis of this condition. Imatinib mesylate (Gleevec) inhibits several protein tyrosine kinases, including platelet-derived growth factor receptor (PDGFR) and c-kit, which are preferentially expressed in tumor cells. We tested the activity of imatinib mesylate in MBC with overexpression of PDGFR or c-kit. Additionally, we sought to determine the biological correlates and immunomodulatory effects. PATIENTS AND METHODS: Thirteen patients were treated with Imatinib administered orally at 400 mg p.o. b.i.d. (800 mg/day), until disease progression. All patients demonstrated PDGFR-beta overexpression and none showed c-kit expression. RESULTS: No objective responses were observed among the 13 patients treated in an intention-to-treat analysis. All patients experienced disease progression, with a median time to progression of 1.2 months. Twelve patients have died, and the median overall survival was 7.7 months. No patient had a serious adverse event. Imatinib therapy had no effect on the plasma levels of the angiogenesis-related cytokines, vascular endothelial growth factor, PDGF, b-fibroblast growth factor, and E-selectin. Immune studies showed imatinib inhibits interferon-gamma production by TCR-activated CD4(+) T cells. CONCLUSION: Imatinib as a single agent has no clinical activity in PDGFR-overexpressing MBC and has potential immunosuppressive effects.

Clinical and experimental findings in humans and animals with chemotherapy-induced peripheral neuropathy.

Pain arises from numerous causes in cancer patients. Well known to cancer care providers, but perhaps less well so to others, is that the main causes of pain in cancer patients in fact arise due to cancer treatments more so than the disease itself. In this paper clinical and laboratory findings on the characteristics of chemotherapy-induced neuropathic pain are reviewed and a scheme for the underlying mechanisms is outlined.

Imatinib mesylate suppresses cytokine synthesis by activated CD4 T cells of patients with chronic myelogenous leukemia.

Although imatinib mesylate (IM) is highly effective at inducing complete cytogenetic remission in patients with chronic myelogenous leukemia (CML), it is known to suppress T-cell proliferation in vitro. As cytokines are required for T-cell proliferation, we investigated the effects of IM on cytokine synthesis by T cells of CML patients by assessing cytokine synthesis by activated CD4+ and CD8+ T cells in vitro. The activation of T cells in the whole blood of IM-treated patients (CML-IM) with Staphylococcus enterotoxin B resulted in significantly lower percentages of CD4+ T cells that synthesized interleukin 2 (P = 0.017), interferon-gamma (P = 0.010), and tumor necrosis factor-alpha (P = 0.009) than did the activated T cells of control subjects. The addition of exogenous IM to the cultures of peripheral blood mononuclear cells of CML-IM patients reduced Th1 cytokine synthesis by the CD4+ T cells. Furthermore, IM therapy at clinical doses suppressed the tyrosine phosphorylation of ZAP70. These findings suggest that inhibition of ZAP70 signaling pathway and suppression of Th1 cytokine synthesis by CD4+ T cells required the presence of IM at the time of T-cell activation through the T-cell receptor.

Soluble TNF-alpha receptor 1 and IL-6 plasma levels in humans subjected to the sleep deprivation model of spaceflight.

BACKGROUND: The extent to which sleep loss may predispose astronauts to a state of altered immunity during extended space travel prompts evaluation with ground-based models. OBJECTIVE: We sought to measure plasma levels of selected cytokines and their receptors, including the putative sleep-regulation proteins soluble TNF-alpha receptor (sTNF-alpha R) I and IL-6, in human subjects undergoing 2 types of sleep deprivation during environmental confinement with performance demands. METHODS: Healthy adult men (n = 42) were randomized to schedules that varied in severity of sleep loss: 4 days (88 hours) of partial sleep deprivation (PSD) involving two 2-hour naps per day or 4 days of total sleep deprivation (TSD). Plasma samples were obtained every 6 hours across 5 days and analyzed by using enzyme-linked immunoassays for sTNF-alpha RI, sTNF-alpha RII, IL-6, soluble IL-2 receptor, IL-10, and TNF-alpha. RESULTS: Interactions between the effects of time and sleep deprivation level were detected for sTNF-alpha RI and IL-6 but not for sTNF-alpha RII, soluble IL-2 receptor, IL-10, and TNF-alpha. Relative to the PSD condition, subjects in the TSD condition had elevated plasma levels of sTNF-alpha RI on day 2 (P =.04), day 3 (P =.01), and across days 2 to 4 of sleep loss (P =.01) and elevated levels of IL-6 on day 4 (P =.04). CONCLUSIONS: Total sleep loss produced significant increases in plasma levels of sTNF-alpha RI and IL-6, messengers that connect the nervous, endocrine, and immune systems. These changes appeared to reflect elevations of the homeostatic drive for sleep because they occurred in TSD but not PSD, suggesting that naps may serve as the basis for a countermeasures approach to prolonged spaceflight.

Interleukin-6 and interleukin-10 levels in chronic lymphocytic leukemia: correlation with phenotypic characteristics and outcome.

The objective of this study was to examine the correlation between serum interleukin-6 (IL-6) and IL-10 levels and outcome in chronic lymphocytic leukemia (CLL). Serum IL-6 and IL-10 levels were measured by enzyme-linked immunoabsorbent assays from 159 and 151 CLL patients, respectively, and from healthy control subjects (n = 55 [IL-6]; n = 37 [IL-10]). Cytokine levels were correlated with clinical features and survival. Serum IL-6 levels were higher in CLL patients (median, 1.45 pg/mL; range, undetectable to 110 pg/mL) than in control subjects (median, undetectable; range, undetectable to 4. 30 pg/mL) (P <.0001). Serum IL-10 levels were higher in CLL patients (median, 5.04 pg/mL; range, undetectable to 74 pg/mL) than in normal volunteers (median, undetectable; range, undetectable to 13.68 pg/mL) (P <.00001). Assays measuring both Epstein-Barr virus-derived and human IL-10 yielded higher values than assays measuring primarily human IL-10 (P <.05). Patients with elevation of serum IL-6 or IL-10 levels, or both, had worse median and 3-year survival (log rank P <.001) and unfavorable characteristics (prior treatment, elevated beta(2)-microglobulin or lactate dehydrogenase, or Rai stage III or IV). Elevated IL-6 and IL-10 levels were independent prognostic factors for survival when analyzed individually or in combination (Cox regression analysis). However, if beta(2)-microglobulin was incorporated into the analysis, it was selected as an independent prognostic feature, and IL-6/IL-10 were no longer selected. In patients with CLL, serum IL-6 and IL-10 (viral and human) levels are elevated and correlate with adverse disease features and short survival. In multivariate analysis, however, beta(2)-microglobulin is the most important prognostic factor.

Restoration of Th1 cytokine synthesis by T cells of patients with chronic myelogenous leukemia in cytogenetic and hematologic remission with interferon-alpha.

Chronic myelogenous leukemia (CML) is a disorder of the hematopoietic stem cell that results in malignant expansion of myeloid cells with a cytogenetic abnormality, the translocation between chromosomes 9 and 22 known as the Philadelphia chromosome. Treatment with IFN-alpha has proven to be an effective therapy, inducing cytogenetic remission in CML patients. However, it is unknown whether IFN-alpha can restore normal immune function for patients who achieve a complete cytogenetic remission. To address this question, we used a method of intracellular staining and flow cytometric analysis to ascribe the syntheses of Th1 or Th2 cytokines to T-cell subsets of patients in chronic, in accelerated, and in blast crisis phases as well as patients who had achieved a complete cytogenetic remission with IFN-alpha. We assessed the cytoplasmic synthesis of cytokine in phorbol ester (phorbol 12-myristate 13-acetate)-activated CD4+ and CD8+ T-cell subsets of 81 patients with various stages of CML and 21 normal controls. The percentages of CD4+ and CD8+ T cells from patients in chronic, in accelerated, and in blast crisis phases that synthesized Th1 cytokines interleukin (IL)-2, IFN-gamma, and tumor necrosis factor-alpha were significantly lower than those of remission patients and normal controls. Conversely, the percentages of CD4+ and CD8+ T cells of patients in chronic, in accelerated, and in blast crisis phases of CML preferentially synthesized the Th2 cytokine IL-10. Patients who achieved a durable complete cytogenetic remission for >2 years without maintenance IFN-alpha therapy restored their preference for a Th1 cytokine profile that is necessary for efficient cytotoxic T-cell function.

Synthesis of IFN-gamma by CD8(+) T cells is preserved in HIV-infected women with HPV-related cervical squamous intraepithelial lesions.

OBJECTIVE: The aim of this study was to investigate whether coinfection with HIV affects the synthesis of Th1 and Th2 cytokines by peripheral blood T cells of women infected with human papillomavirus (HPV). METHODS: Cervical swabs and peripheral blood were obtained from women referred for colposcopy. HPV DNA by Digene's hybrid capture assay, HIV RNA by Roche's Amplicor assay, and cytokine synthesis of T-cell subsets by flow cytometry were assessed. HPV-associated cervical and HIV-associated immune deficiency diseases were staged using the Bethesda System and the Centers for Disease Control criteria, respectively. RESULTS: Patients with HIV and/or HPV infections had lower percentages of IL-2(+) and higher percentages of IL-10(+) T cells than healthy women. Furthermore, women with both virus infections (HIV(+)/HPV(+)) had significantly fewer IL-2(+) CD4(+), IFN-gamma(+) CD4(+), and TNF-alpha(+) CD4(+) T cells than women with HPV infection alone (HPV(+)). Whereas HIV(+) and healthy women had similar numbers of IFN-gamma(+) CD8(+) T cells, HPV(+) women had significantly fewer IFN-gamma(+) CD8(+) T cells than healthy women. CONCLUSION: HIV infection adversely affects the synthesis of Th1 cytokines by CD4(+), but not IFN-gamma synthesis by CD8(+) T cells of women with active HPV infection. The increase in IFNgamma(+) CD8(+) T cells, a phenotype consistent with cytotoxic T lymphocytes, may account for the stable HIV disease of the women studied. However, the increase in IFN-gamma(+) CD8(+) T cells is less likely to be HPV-specific as there was a higher incidence of HPV-related cervical SIL in HIV(+)/HPV(+) women compared with HPV(+) women.

Dysregulated synthesis of intracellular type 1 and type 2 cytokines by T cells of patients with cutaneous T-cell lymphoma.

Mycosis fungoides (MF) and Sezary syndrome (SS) are the two main clinical entities of cutaneous T-cell lymphoma (CTCL). As the disease progresses from MF to SS, a switch from a type 1 (interleukin [IL]-2 and gamma interferon [IFN-gamma]) to a type 2 (IL-4) cytokine production profile occurs. Although roles for type 1 and type 2 cytokines in the pathogenesis of CTCL have been proposed, the cellular origins of these cytokines are unclear. Using flow cytometry to identify individual T-cell subsets, we studied cytokine synthesis by the T cells of 13 patients with SS and 12 with MF and 9 hematologically healthy donors. Upon activation with phorbol 12-myristate 13-acetate (PMA), the numbers of T cells synthesizing IL-2 were similar for all study groups. Whereas the predominant T-cell producing IL-2 in healthy donors and in those with MF was CD7(+), in patients with SS, it was CD7(-). Although the number of IL-4(+) CD4(+) T cells was low for all study groups, there was a significantly higher number of IL-4(+) CD8(+) T cells in patients with MF than in those with SS or healthy donors. There was a decline in the number of IFN-gamma-producing T cells in CTCL donors compared to that in healthy donors. More importantly, there was a significant decrease in the number of IFN-gamma-producing T cells with disease progression from MF to SS. The inability of these T cells to synthesize IFN-gamma may have prognostic value in CTCL, since it may be responsible for the progression of the disease from MF to SS.

Induction of labyrinthitis ossificans after pneumococcal meningitis: an animal model.

Newly formed disorganized bone fills the open spaces within the otic capsule in various pathologic conditions, resulting in labyrinthitis ossificans. The pathologic mechanisms of this disease remain poorly understood. To better study the sequence of events and contributing mechanisms involved in labyrinthitis ossificans, an animal model was developed. Three groups of Mongolian gerbils received either an intralabyrinthine injection of normal saline solution (group 1) or Streptococcus pneumoniae polysaccharide capsule antigens (groups 2 and 3). The temporal bones were harvested after 3 months and serially sectioned. None of the eight control animals (group 1), which received intralabyrinthine injections of normal saline solution had any histologic changes in their temporal bones. Nine of the surviving 19 animals in groups 2 and 3 had fibrosis or evidence of early ossification. A fourth group of Mongolian gerbils received two intrathecal injections of live S. pneumoniae organisms. The temporal bones were harvested after 3 months and serially sectioned. Fourteen of the surviving 15 animals had fibrosis or ossification or both. This animal model will provide a method for study of the mechanisms of labyrinthitis ossificans.

Role of placental cytokines and inflammation in vertical transmission of HIV infection.

In light of new evidence suggesting that maternal human immunodeficiency virus (HIV) infection produces at least a three-fold increase in the number of early spontaneous abortions, it is important to search for factors that may predispose to fetal wastage. Immunological factors are thought to play an important role in permitting the HLA-disparate fetus to continue to term, despite powerful maternal immune forces capable of rejection. In the context of a heightened incidence of spontaneous abortion in HIV infection, evidence is now accumulating that implicates an imbalance in immune factors in contributing to this fetal loss. Soluble immune factors, such as cytokines, have been suggested as contributing agents to recurrent spontaneous abortions. Inflammatory cytokines-interleukin 1beta, interleukin 6 and tumor necrosis factor alpha-have been measured in isolated placental trophoblastic cells in HIV-infected and non-infected pregnant women in an attempt to explore this hypothesis. These inflammatory cytokines and their messenger RNAs were significantly elevated before and after stimulation in HIV-infected women, supporting the belief that HIV-infected women present their fetuses a milieu of imbalanced immune factors capable of contributing to immunological rejection. In addition, these elevated inflammatory cytokine levels may contribute to HIV disease progression in fetuses by virtue of activation of HIV gene transcription factors similar to what has been demonstrated in in vitro systems. We therefore propose that HIV infection in pregnant women produces an altered state of certain soluble immune factors, which in concert with other immune factor abnormalities, such as loss of immune selection in the fetal thymus, predisposes the fetus to advanced HIV infection and possible spontaneous abortion.

Inflammatory cytokine expression is correlated with the level of human immunodeficiency virus (HIV) transcripts in HIV-infected placental trophoblastic cells.

The inflammatory cytokines interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) have been associated with increased human immunodeficiency virus (HIV) expression and enhanced lymphocyte adhesion to trophoblastic cells in experimental systems. To determine if there is a correlation between the expression of these cytokines and the levels of HIV transcripts in trophoblasts of term placentas from HIV-infected women, we studied the placentae of 30 HIV-positive and 13 control gravidae. Twenty-three of the HIV-positive women received zidovudine (ZDV) as prophylaxis against HIV vertical transmission; only one of the seven women who did not receive ZDV was a transmitter, for an overall vertical transmission rate of 3.8%. Cytokine production was measured by enzyme-linked immunosorbent assay in the supernatants of trophoblastic cell cultures. Additionally, cytokine transcripts and HIV gag sequences were determined by a quantitative reverse transcription-PCR assay. In general, trophoblastic cells of HIV-positive placentas expressed significantly higher levels of IL-1beta, IL-6, and TNF-alpha than those of control placentas. All placentas from HIV-positive women expressed HIV gag transcripts at either a low (<156 copies per microg of total RNA) or a high (>156 copies per microg of total RNA) level. There was a statistically significant positive association between the basal level of TNF-alpha production and the level of HIV gag transcripts of HIV-positive placental trophoblastic cells. Nevertheless, these data, coupled with a low transmission rate, would indicate that some other factors, perhaps working in concert with cytokines, are necessary for vertical transmission of HIV from mother to infant.

Induction of inflammatory cytokines in placental monocytes of gravidae infected with the human immunodeficiency virus type 1.

Placental mononuclear cells (PMC) are susceptible to infection with the human immunodeficiency virus (HIV). PMC secreted tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta), and IL-6 among other factors, which, in turn, regulate HIV replication in latently infected cells. We assessed the induction of these cytokines in PMC from HIV-infected (HIV+) and uninfected (control) gravidae following exposure to lipopolysaccharide (LPS), HIV lysate (iHIV), recombinant HIV env (GP160) and HIV gag (gag55), and synthetic HIV p17 (HGP30) antigens. In comparison to control PMC, HIV+ PMC constitutively secreted higher levels of IL-1beta and IL-6 and were refractory to stimulation by iHIV, GP160, gag55, and HGP30. Control PMC IL-1 beta levels were boosted by LPS; gag55 and HGP30 augmented IL-6 but not IL-1 beta. Both groups exhibited low basal TNF-alpha production that was augmented by LPS. HIV+ PMC exhibited higher constitutive levels of IL-1 beta, IL-6, and TNF-alpha gene transcription than control PMC. These levels could be further augmented by LPS, yet the incremental levels were lower than those obtained from PMC of uninfected women. The high basal constitutive secretion of cytokines by HIV+ PMC and their refractoriness to activation may reflect a virus-mediated dysregulation of cytokine expression culminating in compromised host defenses against secondary opportunistic infections associated with AIDS.

Epidermal growth factor expression in human colon and colon carcinomas: anti-sense epidermal growth factor receptor RNA down-regulates the proliferation of human colon cancer cells.

Human colon cancer cell lines express epidermal growth factor (EGF) mRNA, secrete EGF and may respond to it via the cell-surface EGF receptor (EGFR). Expression of these molecules in human colon and colon tumor, however, is not clear. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of RNA prepared from paired normal human colon and colon tumor samples from 12 individuals followed by Southern blotting analyses of the RT-PCR products revealed a major fragment of 527 bp and a minor fragment of 404 bp that hybridized to a human EGF cDNA probe under stringent conditions. Identical results were obtained from 8 human colon cancer cell lines. Cloning and sequencing of PCR products confirmed that both fragments were from the human EGF gene; the 527-bp fragment corresponded exactly to nucleotides 2,891 to 3,417 of the human EGF mRNA reported by others. A deletion of 123 nucleotides (nucleotides 3,172 to 3,294) was found in the 404-bp fragment. Immunohistochemical studies using cyostat sections of human colon specimens showed that EGF was expressed in the human colon and that expression was restricted to the epithelial colonic crypt cells and epithelium-derived cancer cells. Since EGF and EGF-related molecules are potent mitogens that mediated their effect through the EGFR, we also determined the efficacy of anti-sense EGFR RNA in circumventing the EGFR-related pathway of proliferation. Expression of anti-sense EGFR RNA, by transfection with an inducible anti-sense EGFR expression vector, down-regulated cell-surface EGFR expression and proliferation of these cells and their ability to grow in soft agar. Anti-sense EGFR RNA was found to be an anti-proliferative agent in both relatively non-aggressive and highly aggressive human colon cancer cells.

Overexpression of flbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brlA and premature initiation of development.

Aspergillus nidulans reproduces asexually by forming thousands of mitotically derived spores atop highly specialized multicellular organs termed conidiophores. We have identified a gene called flbA (for fluffy low brlA expression) that is required for initiation of A. nidulans conidiophore development. flbA mutants form abnormal colonies that have a distinct fluffy phenotype characterized by tightly interwoven aerial hyphae that autolyse as the colony matures. The requirement for flbA in conidiophore development precedes activation of brlA, a primary regulator of conidiophore development. The wild-type flbA gene was isolated and found to encode a 3.0 kb mRNA that is expressed throughout the A. nidulans asexual life cycle. Overexpression of flbA using an inducible promoter resulted in misscheduled expression of brlA in vegetative cells and caused hyphal tips to differentiate into spore-producing structures. Sequence analysis of a nearly full-length flbA cDNA clone showed that flbA is predicted to encode a 717-amino-acid polypeptide with 30% identity to the Saccharomyces cerevisiae SST2 protein. SST2 is required by yeast cells for resuming growth following prolonged exposure to yeast mating pheromone and for mating partner discrimination. We propose that flbA plays a related role in a signalling pathway for Aspergillus conidiophore development.

The Aspergillus nidulans fluG gene is required for production of an extracellular developmental signal and is related to prokaryotic glutamine synthetase I.

Mutations in the Aspergillus nidulans fluG gene disrupt the programmed induction of asexual sporulation and result in formation of fluffy colonies that are characterized by undifferentiated cotton-like masses of vegetative cells. We show that the fluG mutant phenotype is suppressed when fluG mutant colonies are grown next to wild-type colonies even if the two strains are separated by dialysis membrane with a 6000- to 8000-dalton pore size. fluG encodes a cytoplasmically localized approximately 96,000-dalton polypeptide that is present at relatively constant levels during vegetative growth and following developmental induction. Sequence analysis of fluG demonstrated that the carboxy-terminal 436 amino acids predicted by the 864-codon FluG open reading frame shares approximately 28% identity with GSI-type prokaryotic glutamine synthetases. We consider it unlikely that FluG functions in synthesis of glutamine but instead propose that FluG functions as a GSI-related enzyme in synthesizing an extracellular signal directing asexual sporulation and perhaps other aspects of colony growth. The relationships between fluG and other genes identified by fluffy mutants are discussed.