Specific interactions between Smad proteins and AP-1 components determine TGFß-induced breast cancer cell invasion.

Deregulation of the transforming growth factor ß (TGFß) signal transduction cascade is functionally linked to cancer. In early phases, TGFß acts as a tumor suppressor by inhibiting tumor cell proliferation, whereas in late phases, it can act as a tumor promoter by stimulating tumor cell invasion and metastasis. Smad transcriptional effectors mediate TGFß responses, but relatively little is known about the Smad-containing complexes that are important for epithelial-mesenchymal transition and invasion. In this study, we have tested the hypothesis that specific members of the AP-1 transcription factor family determine TGFß signaling specificity in breast cancer cell invasion. Using a 3D model of collagen-embedded spheroids of MCF10A-MII premalignant human breast cancer cells, we identified the AP-1 transcription factor components c-Jun, JunB, c-Fos and Fra1 as essential factors for TGFß-induced invasion and found that various mesenchymal and invasion-associated TGFß-induced genes are co-regulated by these proteins. In situ proximity ligation assays showed that TGFß signaling not only induces complexes between Smad3 and Smad4 in the nucleus but also complexes between Smad2/3 and Fra1, whereas complexes between Smad3, c-Jun and JunB could already be detected before TGFß stimulation. Finally, chromatin immunoprecipitations showed that c-Jun, JunB and Fra1, but not c-Fos, are required for TGFß-induced binding of Smad2/3 to the mmp-10 and pai-1 promoters. Together these results suggest that in particular formation of Smad2/3-Fra1 complexes may reflect activation of the Smad/AP-1-dependent TGFß-induced invasion program.

Myelin protein P0-specific IgM producing monoclonal B cell lines were established from polyneuropathy patients with monoclonal gammopathy of undetermined significance (MGUS).

Monoclonal expansion of B cells and plasma cells, producing antibodies against 'self' molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenström's macroglobulinaemia and B-type of chronic lymphocytic leukaemia (B-CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN-MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin-specific B cell clones from peripheral blood of patients with PN-MGUS, by selection of cells bearing specific membrane Ig-receptors for myelin protein P0, using beads coated with P0. P0-coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein--Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti-P0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5+ positive, although the expression declined in vitro over time. The anti-P0 antibodies were of IgM-lambda type. The antibodies belonged to the VH3 gene family with presence of somatic mutations. The IgM reacted with P0 and myelin-associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5+ clones included in the study as controls. The expanded clones expressed CD80 and HLA-DR, which is compatible with properties of antigen-presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P0-specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen-presentation by these cells followed by T cell activation.

Long-term results in otosclerotic patients operated by stapedectomy or stapedotomy.

Stapedectomy and stapedotomy are the techniques currently used in the surgical treatment of otosclerosis. During the period 1977-81, a total of 60 consecutive patients were subjected to surgery for otosclerosis according to the method of Schuknecht (Gelfoam and wire prosthesis). Another 55 consecutive otosclerotic patients were operated on during 1987-91with stapedotomy, according to the method advocated by Fisch (Fisch-type piston). Independent of the surgical technique used, the maximal hearing gain was obtained 1 year after surgery. In the frequency range 0.5-3 kHz, the mean value of the averaged pure tone thresholds improved from 52 to 28 dB in the stapedectomy group and from 57 to 26 dB in the stapedotomy group. In the frequency range 4-6 kHz, the subjects' hearing was not significantly improved in the stapedectomy group, whereas the subjects' hearing in the stapedotomy group improved from 61 to 44 dB. Per- and postoperative complications were low in both groups, except for one patient in the stapedectomy group who experienced the serious complication of a deaf ear after surgery.

Thioredoxin prolongs survival of B-type chronic lymphocytic leukemia cells.

Thioredoxin (Trx) is a ubiquitous protein disulfide oxidoreductase with antioxidant, cytokine, and chemotactic properties. Previously, we showed that Trx, in synergy with interleukin 1 (IL-1), IL-2, IL-4, tumor necrosis factor alpha (TNF-alpha), and CD40-ligation induced S-phase entry and mitosis in normal B cells and B-type chronic lymphocytic leukemia (B-CLL) cells. The viability of B-CLL cells stimulated by these protocols is high, and it has been hypothesized that the overexpression of Bcl-2 found in B-CLL protects the cells from apoptosis in vitro and in vivo. In this study, we have analyzed the response of cells derived from 12 samples of patients with B-CLL to recombinant human Trx in spontaneous apoptosis, with special reference to the Bcl-2 expression. Long-term cultures of B-CLL clones showed significantly higher viability when supplemented with human Trx (P =.031), also exemplified with clones surviving more than 2 months. Short-term cultures of B-CLL cells exposed to 1 microg/mL of Trx for 1, 5, or 12 days maintained expression or delayed down-regulation of Bcl-2 compared with control cultures containing RPMI 1640 medium and 10% fetal calf serum only (P =.032,. 002,.026, respectively). All B-CLL cells expressed constitutive Trx at varying but low levels, in contrast to adult T-cell leukemias, which overexpress Trx, as previously reported. We found that Trx added to B-CLL cells increased in a dose-dependent fashion the release of TNF-alpha, which has been suggested to be an autocrine growth factor for these cells. In conclusion, we have found that human recombinant Trx induced TNF-alpha secretion, maintained Bcl-2, and reduced apoptosis in B-CLL cells. (Blood. 2000;95:1420-1426)

Tele-otolaryngology consultations between two rural primary-care centres in southern Lapland and the University Hospital of Umeå.

In 1996 a telemedicine link was established between two primary-care centres of Västerbotten county and the University Hospital. Specialties involved at the University Hospital were otoloaryngology, orthopaedics and dermatology. Videoconferencing used ISDN at 384 kbit/s. The primary-care centres were equipped with video-endoscopes. During the first 21 months, there were 32 otolaryngology consultation. The average time for each consultation was between 15 and 30 min. Patients, general practitioners and specialists were interviewed using questionnaires with answers on a six-point scale, in which a score of six was best. Patient satisfaction produced a mean score of 5.7. The specialist doctors rated the video-consultation satisfactory for diagnosis. Roughly 40% of the referrals could be avoided by telemedicine. The general practitioners rated the educational effect of the consultation very highly.

Staphylococcus aureus Cowan strain 1 activation of B-chronic lymphocytic leukaemia cells augments the response to CD40 stimulation.

The signals involved in regulating the proliferation, differentiation and survival of B-chronic lymphocytic leukemia (B-CLL) cells are fully understood. B-CLL cells have been found to respond poorly to various activation signals and only after successful Epstein-Barr virus (EBV) transformation has it been possible to maintain such cells in long-term cultures. In this work we describe a new method to activate and induce proliferation in B-CLL cells and to maintain such cells in long-term culture for longer than 1 month. We used a combination of protocols in an attempt to mimic some of the signals of a thymus-dependent immune response. The B-CLL cells were first activated with Staphylococcus aureus Cowan strain 1 (SAC) particles plus thioredoxin (Trx), followed by stimulation with interleukin (IL)-2 + Trx. This treatment primed the cells for further stimulation with anti-CD40 monoclonal antibody (MoAb) presented on irradiated CD32L cells (the CD40-system) or soluble CD40 Ligand, and a combination of Trx and cytokines (IL-4 + IL-10), which allowed the cells to be maintained for up to 1 month with preserved viability and a variable rate of proliferation. However, induced proliferation of the B-CLL cells was limited to approximately 1 month, suggesting that additional signals are required to facilitate further proliferation.

Growth and survival of B-chronic lymphocytic leukaemia cells.

Although chronic lymphocytic leukaemia of B cell type (B-CLL) is the most common form of leukaemia in the Western world, several questions about the biology of B-CLL remain to be clarified. To obtain a conceptual model for B-CLL, defined as a relentless accumulation of resting B-CLL cells, it is particularly relevant to ask which cell type is the normal counterpart of B-CLL; what is the site of proliferation; which signals are involved in the recruitment and induction of proliferation and which signals contribute to the survival of the B-CLL cells? The significance of the studies on B-CLL cells in vitro for the interpretation of the in vivo situation may be questioned since they oversimplify the multiple and complex cellular interactions that occur in vivo. However, the in vitro studies have been instrumental in elucidating signals that may regulate growth, differentiation and survival of B-CLL cells. This knowledge, herein reviewed, can be used to put forward a hypothesis on B-CLL cell regulation in vivo.

Interleukin-15 + thioredoxin induce DNA synthesis in B-chronic lymphocytic leukemia cells but not in normal B cells.

We have previously shown that Staphylococcus aureus Cowan strain 1 particles (SAC) + thioredoxin (Trx) + IL-2 may induce B-chronic lymphocytic leukemia (B-CLL) cells to proliferate. In this paper we have examined IL-15, which has activities similar to IL-2, for its ability to stimulate B-CLL cells and compared its activity with that of IL-2. We found that B-CLL cells could be induced to DNA synthesis upon treatment with IL-15 + Trx. The presence of Trx was essential for the IL-15-induced DNA synthesis. This contrasts to the effect of IL-15 + Trx on normal CD5+ and CD5- B cells, where IL-15 + Trx alone only induced limited DNA synthesis. IL-15 was as effective in the induction of DNA synthesis in B-CLL cells as IL-2, but about 100-fold less potent with an EC50 of 200 ng/ml. In addition we found that the IL-15 + Trx-induced proliferation was inhibited by CD40 stimulation. We conclude that IL-15 together with a proper costimulus can induce B-CLL cells to proliferate in vitro.

The effects on growth and survival of IL-6 can be dissociated in the U-266-1970/U-266-1984 and HL407E/HL407L human multiple myeloma cell lines.

Several studies have documented IL-6-dependent growth promotion of murine and human neoplastic plasma cells. However, it is well known that human multiple myeloma (MM) cells in vitro show a considerable degree of heterogeneity concerning growth and survival requirements. This heterogeneity, which probably reflects overlapping effects of feeder cells, interleukin 6 (IL-6) and components of fetal calf serum (FCS) as well as tumour heterogeneity in vivo, has hampered the elucidation of molecular mechanisms underlying the effects of IL-6. In an attempt to dissociate growth and survival promotion of IL-6, we have studied two pairs of human MM cell lines, HL407E/HL407L and U-266-1970/U-266-1984, selected to represent different stages of in vitro tumour progression and dependence of feeder cells and exogenous IL-6. We demonstrated that exogenous IL-6, in the presence of FCS, conveyed: (a) a strong growth stimulatory effect with weak or no survival promotion in HL407L and U-266-1970 cells; (b) promotion of survival with no effects on growth in HL407E cells; (c) no growth or survival promotion to U-266-1984. Moreover, our results suggested that IL-6 may enhance apoptosis in U-266-1970/U-266-1984 cells, and that FCS may interfere with IL-6 in its growth stimulatory effect. The relative dissociation of growth, survival and apoptotic effects of IL-6 leads to the conclusion that the HL407E/HL407L and U-266-1970/U-266-1984 pairs of cell lines provide a useful human model system to study molecular mechanisms underlying these separate events.

CD40 ligation inhibits IL-2 and SAC+IL-2 induced proliferation in chronic lymphocytic leukaemia cells.

This paper reports on differences between B cell-type chronic lymphocytic leukaemia (B-CLL) and normal B cells in their response to IL-2+thioredoxin (Trx), Staphylococcus aureus Cowan strain 1 (SAC)+IL-2+Trx, and to CD40 ligation of IL-2+Trx and SAC+IL-2+Trx stimulation. The authors found that Trx acted synergistically with IL-2 and SAC+IL-2 in inducing DNA synthesis in B-CLL cells, but not in the normal B cells. Interestingly, IL-2+Trx alone was found to induce proliferation in B-CLL cells from patients with advanced stages of disease. In addition, we also found that IL-2+Trx and SAC+IL-2+Trx-induced DNA synthesis of B-CLL cells was inhibited by CD40 activation (by soluble anti-CD40 MoAb and anti-CD40 MoAb presented on irradiated CD32L cells). In clear contrast, SAC+IL-2+ Trx-induced DNA synthesis of normal B cells was not inhibited by soluble anti-CD40 MoAb. The authors therefore conclude that B-CLL cells differ from normal B cells in their response to IL-2 (IL-2+Trx) and CD40 ligation.

Interleukin-4 (IL-4) enhances homotypic adhesion of activated B-chronic lymphocytic leukaemia (B-CLL) cells via a selective up-regulation of CD54.

It is well established that cell-to-cell contact modifies cytokine signalling but little is known on the role of homotypic cell adhesion for proliferation and differentiation of B cells. Homotypic adhesion involves mainly the interaction between the adhesion molecules Leukocyte Function Antigen-1 (LFA-1) and its ligand CD54 (ICAM-1). A well-characterized B-chronic lymphocytic leukaemia (B-CLL) clone (I-83) was used as a source of monoclonal B cells inducible to DNA synthesis and differentiation by using 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in combination with interleukin-4 (IL-4) and thioredoxin (Trx)-containing supernatant from a T-cell hybridoma (BSF-MP6). This paper shows that IL-4 alone was able to induce aggregation of B-CLL cells and to strongly enhance TPA+BSF-MP6-induced aggregation. The results from studying the expression of CD11a and CD18, the two subunits of LFA-1, and CD54 during stimulated DNA synthesis and differentiation suggest that IL-4-induced, or enhanced, aggregation was mainly mediated by a selective up-regulation of CD54. It was further demonstrated by antibody blockade to either CD11a, CD18 or CD54 that aggregation could be inhibited without affecting induced DNA synthesis or differentiation.

A comparison of hyaluronan and fibronectin in the healing of tympanic membrane perforations.

Fibronectin and 1% hyaluronan, two extracellular matrix components, were applied to tympanic membrane (TM) perforations, using the laboratory rat as an animal model. The perforations occupied the upper, posterior quadrant of the TM. Saline-treated and untreated perforations of equal size served as controls. Fifty percent of the perforations treated daily with hyaluronan healed prior to the first closures of the other groups. The use of fibronectin did not enhance the healing rate. The hyaluronan-treated perforations were covered initially by a sheet of keratin and hyaluronan containing abundant inflammatory cells. Within this keratin-hyaluronan cover, hyperplastic stratified keratinizing epithelium advanced and bridged the gap of the perforation ahead of the approaching connective tissue. These findings indicate that exogenously applied hyaluronan could be valuable in the clinical situation and should be tried to improve the healing of TM perforations.

A case of a double tongue. Case report.

A baby boy presented with a double tongue combined with a median cleft palate. He was otherwise normally developed. The double tongue was reconstructed to form one tongue 40 days after birth. At follow-up examination when he was 6 months old the tongue was well formed with normal function.

Repair of chronic tympanic membrane perforations using applications of hyaluronan or rice paper prostheses.

A controlled randomized study was performed in 60 patients with 64 chronic, dry tympanic membrane (TM) perforations. The perforations were randomly allocated to either resection of the perforation rim and instillation of 1% hyaluronan (Healon; HYA) in the perforation gap once daily for 7 days (33 ears) or resection of the perforation margin and application of a sterile rice paper prosthesis (31 ears). The treatment effect was documented by TM photography and morphometric measurements of the perforation area. The hearing was assessed with puretone and high-frequency audiometry. After 2 months, 5 of the HYA-treated perforations (15%) and 4 of the rice-paper-treated TMs (13%) were healed. After 1 year, 18 perforations (9 in each treatment group) were healed. In neither group were there any persistent adverse effects on hearing. It is noteworthy that 28% (18/64) of the chronic, long-standing TM perforations could be repaired by these technically simple and time-saving methods. Both procedures should be considered as easy first-choice alternatives to myringoplasty in selected cases.

Consequences of using hyaluronan-coated tympanostomy tubes.

The effects of hyaluronan-coated polyethylene tympanostomy tubes on the tympanic membrane structure were investigated and compared with non-coated polyethylene tubes without the coating. The tubes were inserted into the tympanic membrane (TM) and removed 3 weeks later. After 3 weeks the tubes were reinserted. This tube insertion and removal and reinsertion sequence was repeated 4 times and the tympanic membrane structure was examined histologically at 3 weeks and 12 weeks later. The study did not reveal any differences regarding the thickness and light microscopical appearance when comparing the TMs with the hyaluronan-coated tubes to those with the non-coated polyethylene tubes. However, the present way of evaluating the effects of different tube materials may be too crude. In future experiments aimed at elucidating the effects of different surface coatings of tympanostomy tubes other experimental designs must be employed, e.g. fewer tube insertion sequences and less traumatizing extractions.

Structural changes in the tympanic membrane after repeated tympanostomy tube insertion.

In an animal model (rat) a polyethylene tympanostomy tube was repeatedly inserted (four periods lasting 2 weeks) into the upper rear quadrant of the right tympanic membrane (TM). The intervals between the different tubulation periods (TPs) lasted 3 weeks. The corresponding quadrant of the left TM was subjected to repeated myringotomies (four times). The structural changes in the TMs were evaluated otomicroscopically and by histological techniques 3 weeks and 3 months after the final TP. Repeated tympanostomy tube insertion caused a dramatically thickened pars tensa. The thickened areas were characterized by a scar tissue exhibiting sclerotic plaques and a dense connective tissue with bone-like formations. Occasionally, islands of keratinizing stratified squamous epithelium were noted within the thickened pars tensa as well as interrupting the epithelial lining facing the tympanic cavity. Similar structural changes occurred after myringotomy without tube insertion, but they were not so pronounced as after repeated tympanostomy tube insertion. The changes were not restricted to the manipulated quadrants, but also affected the untouched anterior quadrants. Throughout the observation period the anterior quadrants improved, while the rear quadrants remained severely affected.

Tympanostomy tubes for prevention of purulent otitis media.

A recently developed animal model was used to study the effect of tympanostomy tubes (TTs) on the spontaneous development of purulent otitis media. In 35 rats with soft-palate clefts a TT was inserted into the right tympanic membrane. The left ear was left intact. Serous effusion occurred in the attic space within 2 days after surgery, whether or not the middle ear cavity (MEC) was artificially ventilated. Between days 7 and 21 the intact-ear MEC was gradually filled with effusion material that turned purulent. Effusion material did not develop in the mesotympanum and hypotympanum of the intubated ears. Microbiologic examination of the effusion material showed a microflora similar to that in the nasopharynx. Ventilation through a TT reduced the number of colonized MECs (4 vs. 10) on day 21. In the individual culture-positive MEC with a TT there were fewer colonies than in the corresponding ear without a TT. These results support the contention that a TT may prevent the development of purulent otitis media.