Tumor necrosis factor alpha interferes with the cell cycle of normal and papillomavirus-immortalized human keratinocytes

We have shown that normal and human papillomavirus (HPV) type 16 immortalized human foreskin keratinocytes are growth inhibited by tumor necrosis factor alpha (TNF-alpha), whereas HPV-18- and SV40-immortalized keratinocytes are resistant to this cytokine (1). In this report, we investigated the expression of mitotic regulatory proteins, such as cyclin A, cyclin B, and p34cdc2. After exposure to TNF-alpha, normal and HPV-16-immortalized cells exhibited a dramatic decrease in the expression of these proteins. In contrast, no alteration in the levels of these proteins was observed after treatment of the resistant cell lines, as well as two HPV-positive cervical carcinoma cell lines. Expression of cyclin E does not seem to be modulated by TNF-alpha in any of the cells tested. On the other hand, cyclin D1, expression is slightly increased in normal keratinocytes and in the HPV-16-immortalized cells, whereas no alteration was observed in the HPV-18-transfected cells. The phosphorylation state of pRb correlated with cell growth; sensitive cells, which accumulate in G0-G1, after exposure to TNF-alpha, exhibited an accumulation of hypophosphorylated pRb, whereas no effect on pRb phosphorylation was observed for HPV-18-immortalized cells. These results clearly correlate with TNF-alpha-induced growth arrest in G0-G1.     

Amifostine (WR-2721) shortens the engraftment period of 4-hydroperoxycyclophosphamide-purged bone marrow in breast cancer patients receiving high-dose chemotherapy with autologous bone marrow support

4-Hydroperoxycyclophosphamide (4-HC), a commonly used marrow-purging agent, is active against many tumors, but is also toxic to normal marrow progenitors. Amifostine (WR-2721) is a sulfhydryl compound with chemoprotectant activity. Preclinical studies using suspensions of bone marrow and breast cancer cells demonstrated that ex vivo treatment with amifostine followed by 4-HC resulted in protection of marrow progenitors, with no compromise in the antitumor effect of 4-HC. This fact stimulated the development of a clinical trial. Bone marrow was harvested from 15 poor-prognosis breast cancer patients and randomly assigned to ex vivo treatment with amifostine followed by 4-HC (amifostine + 4-HC), or treatment with 4-HC alone. High-dose chemotherapy was then administered followed by infusion of the purged autologous bone marrow support (ABMS). Leukocyte engraftment, defined as a white blood cell count > or = 1 x 10(9)/L, was achieved in an average of 26 days for patients whose marrow was purged with amifostine + 4-HC versus 36 days for patients whose marrow was purged with 4-HC alone (P = .032). The average number of platelet transfusions (12 v 29; P = .017) and days of antibiotic therapy (28 v 40; P = .012) were significantly less for patients whose marrow was exposed to amifostine + 4-HC, compared with 4-HC alone. Unpurged backup marrow fractions were infused into three patients whose marrow was purged with 4-HC alone, because of inadequate marrow recovery. None of the patients who received amifostine + 4-HC-purged marrow required a backup marrow fraction. Complete remissions were achieved in 83% of patients with measurable disease, with no difference between the two cohorts. Forty-three percent of patients remained alive and progression-free at a mean of 13 months posttransplant. There was no significant difference in the rate or pattern of relapse for patients whose marrow was purged with amifostine + 4-HC compared with those whose marrow was purged with 4-HC alone. Ex vivo treatment of marrow with amifostine significantly shortens the time to marrow recovery, thereby reducing the risk of myelosuppressive complications in breast cancer patients receiving high-dose chemotherapy and 4-HC-purged ABMS. Since supportive care requirements are also significantly decreased, amifostine may reduce the cost of such therapy.     

Recovery of gut-associated lymphoid tissue and upper respiratory tract immunity after parenteral nutrition

OBJECTIVE: The authors characterize the recovery of parenteral nutrition-induced changes in gut-associated lymphoid tissue (GALT) and upper respiratory tract immunity with enteral nutrition and provide further information defining the effects of enteral feeding on mucosal immunity. SUMMARY BACKGROUND DATA: The small intestine plays a prominent role in development and maintenance of mucosal immunity, both intestinal and extraintestinal, primarily through immunoglobulin A (IgA)-mediated mechanisms. Prior research has shown that mice fed total parenteral nutrition (TPN) have reduced GALT T and B cells, the cells responsible for IgA production, as well as impaired upper respiratory tract immunity to viral challenge of previously immunized animals. The recovery of TPN-induced changes in GALT and upper respiratory tract immunity after enteral refeeding is studied. METHODS: Male institute of Cancer Research mice received 5 days of TPN followed by 0 to 4 days of chow. Small intestinal GALT was characterized by flow cytometry. In a second experiment, animals were immunized intranasally with moused-adapted influenza virus. Three weeks later, one group received a 5-day course of TPN followed by enteral refeeding for 5 days. A second group received TPN alone. Both groups were challenged with intranasal virus and killed 40 hours postchallenge to determine viral shedding from the upper respiratory tract. RESULTS: Animals fed TPN only had significantly fewer GALT lymphocytes compared with those chow-fed control subjects. Peyer's patch counts increased after a single day of refeeding, returning to normal levels by 48 hours. Lamina propria counts remained significantly depressed after 24 hours of refeeding, but also returned to normal after 48 hours of refeeding. The T-cell and B-cell populations mimicked total cell patterns. Lamina propria CD4+/CD8+ ratio returned to normal only after 72 hours of refeeding. None of the 9 animals refed enterally for 5 days were positive for viral shedding, compared with 8 of 12 matched TPN-fed animals. CONCLUSIONS: Enteral refeeding after TPN is associated with rapid repletion of GALT cellularity, initially within Peyer's patches and subsequently within the lamina propria. Refeeding corrects the impairment of IgA-mediated upper respiratory tract antiviral immunity occurring with TPN administration. This work further enhances the authors' knowledge of the underlying immunologic differences influenced by routes of nutrition.     

A stability criterion for asynchronous multirate linear systems

A stability criterion is developed for linear, asynchronous multirate sampled-data systems. Such systems typically include a linear, time variant, continuous-time plant and several unsynchronized digital controllers. The analysis method uses a time-domain approach based on the closed-loop state transition matrix. The result is sufficient stability criterion which gives an objective measure of long-term and short-term stability. A simple example illustrates the method     

Fade duration analysis on earth-space paths at Ku-band in Fukuoka, Japan

Presented are the fade duration dynamics as observed in four close stations. Fade duration statistics derived from the collected received signal during a critical period in the region are presented and analysed. The obtained results are compared with the model release by the ITU-R P.1623 recommendation. Some modifications are then proposed.     

Requirement of cysteine-rich repeats of the Fas receptor for binding by the Fas ligand

The Fas receptor is a member of a family of cell death receptors, including tumor necrosis factor receptor I (TNFR I), death receptor 3 and 4 (DR3 and DR4), and cytopathic avian receptor 1 (CAR1). The Fas receptor is composed of several discrete domains, including three cysteine-rich domains (CRDs), a transmembrane domain, and an intracellular domain responsible for transmitting an apoptotic signal. While the mechanism of Fas-mediated cell death has become elucidated, the requirements for Fas ligand binding to the receptor have not been fully defined. Using a series of chimeric Fc-receptor fusion proteins between the human Fas receptor and TNFR I, each cysteine-rich domain of Fas was found to be required for interaction with the Fas ligand. Interestingly, TNFR I CRD1 could partially substitute for the Fas CRD1. The importance of this domain was underscored by the analysis of a Fas extracellular mutation (C66R), which resulted in a complete loss of ligand binding. This mutation was cloned from a human patient suffering from Canale-Smith syndrome, which is characterized by autoimmunity resembling that observed in the lpr and lprcg mice. The localization of essential ligand binding domains in the Fas receptor correlated exactly with the ability of the Fas receptor fusion proteins to prevent cell death mediated by the Fas ligand.     

Plasma and blood lead concentrations, lead absorption, and lead excretion in nonhuman primates

The effects of pH, temperature, block of energy production, calcium/calmodulin, protein phosphorylation, and cytoskeleton-disrupting agents (cytochalasin D, nocodazole) on the integrity of the membrane skeleton were studied in polarized MDCK cells. The intracellular distributions of alpha-fodrin, actin, and ankyrin were monitored by immunofluorescence microscopy. The membrane skeleton, once assembled, seemed to be quite stable; the only factors releasing alpha-fodrin from the lateral walls were the acidification of the cytoplasm and the depletion of extracellular calcium ions. Upon cellular acidification, some actin was also released from its normal location along the lateral walls and was seen in colocalization with alpha-fodrin in the cytoplasm, whereas ankyrin remained associated with the lateral walls. No accumulation of plasma membrane lipids was observed in the cytoplasm of acidified cells, as visualized by TMA-DPH. These results suggest that the linkages between the fodrin-actin complex and its membrane association sites are broken upon acidification. The pH-induced change in alpha-fodrin localization was reversible upon restoring the normal pH. Reassembly of the membrane skeleton, however, required temperatures above +20 degrees C, normal energy production, proper cell-cell contacts, and polymerized actin. Release of alpha-fodrin from the lateral walls to the cytoplasm was also observed upon depletion of extracellular calcium ions. This change was accompanied by the disruption of cell-cell contacts, supporting the role of proper cell-cell contacts in the maintenance of the membrane skeleton polarity. These results suggest that local alterations of the cytoplasmic pH and calcium ion concentration may be important in regulating the integrity of the membrane skeleton.     

Identification of an interaction between the m-band protein skelemin and beta-integrin subunits. Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells

Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules. To identify molecules interacting with the cytoplasmic domain of the beta3-integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system. Two identical clones coding for a 96-amino acid sequence were identified. This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle. Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle. The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin. A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with beta1- and beta3-integrin cytoplasmic domains expressed as glutathione S-transferase (GST) fusion proteins, but not with GST-beta2-integrin cytoplasmic tail or GST alone. The skelemin-binding region was in the membrane proximal cytoplasmic domains of the integrins. Full-length skelemin interacted with integrin in intact cells as demonstrated by the colocalization of hemagglutinin-tagged skelemin in Chinese hamster ovary (CHO) cells containing alphaIIbbeta3-integrin and by the finding that microinjection of C2 motif 4 of skelemin into C2C12 mouse myoblast cells caused spread cells to round up. A skelemin-like protein was detected in CHO cells, endothelial cells, and platelets, and this protein colocalized with beta1- and beta3-integrins in CHO cells. This study suggests the presence of a skelemin-like protein in non-muscle cells and provides evidence that it may be involved in linking integrins to the cytoskeleton.