Treatment of cervical intraepithelial neoplasia levels 2 and 3 with adapalene, a retinoid-related molecule.

OBJECTIVES: This study examined dose scheduling, safety, and efficacy of adapalene in the treatment of CIN2 and CIN3. METHODS: Patients were instructed on insertion and removal of an adapalene delivery system. Treatment regimens of 4, 8, and 14 days were utilized. Biopsies were performed on day 90 to assess efficacy. Safety was evaluated with toxicity questionnaires and patient interviews. RESULTS: Two patients treated for 4 days had stable disease. Twenty-three patients treated for 8 days demonstrated an overall 61% (14 of 23) response rate. Twenty-four patients treated for 14 days had an overall 38% (9 of 24) response rate. No patient had disease progression. Compared to untreated historical controls, significantly improved efficacy was demonstrated for patients with CIN2. Patients with CIN3 had improved efficacy, though not statistically significant. CONCLUSIONS: The lack of side effects and practicality of home use make adapalene a nontoxic and safe alternative to surgical therapy in patients with CIN2 and CIN3.

Secretion of thermostable DNA polymerase using a novel baculovirus vector.

Baculovirus-mediated expression has become a powerful tool for the high yield production of functionally active recombinant proteins. In order to further enhance the utility of this expression system, we constructed versatile transfer vectors that facilitate the secretion of recombinant proteins from host insect cells by inserting functional secretory leader sequences down-stream of the polyhedrin promoter. In-frame insertion of cDNA sequences results in the synthesis of fusion proteins containing a heterologous signal sequence which directs the recombinant protein to the secretory pathway. Human and insect leader sequences were successfully tested with a number of proteins including the thermostable Pyrococcus furiousus (Pfu) DNA polymerase in an effort to maximize secretion of heterologous proteins from insect cells. The human placental alkaline phosphatase signal sequence (MLGPCMLLLLLLLGLRLQLSLG) proved to be optimal for the secretion of not only this thermostable bacterial enzyme but also for the secretion of other biologically active polypeptides.

Inhibition of Epstein-Barr virus-mediated capping of CD21/CR2 by alpha interferon (IFN-alpha): immediate antiviral activity of IFN-alpha during the early phase of infection.

Early events of human B-lymphocyte infection by Epstein-Barr virus involve the virus binding to CD21, capping, and subsequent internalization of the virus-receptor complex. We show here that alpha interferon (IFN-alpha) inhibits the capping of Epstein-Barr virus-CD21 complexes. Synthetic peptides with the CD21 binding motif of IFN-alpha mimic IFN-alpha activity, suggesting that this effect may be mediated by IFN-alpha-CD21 interaction. Our findings demonstrate a novel and immediate mechanism of IFN-alpha action.

SrfI, a new type-II restriction endonuclease that recognizes the octanucleotide sequence, [sequence: see text].

A new restriction endonuclease, SrfI has been isolated from an unidentified species of Streptomyces. SrfI recognizes the 8-bp palindrome, 5'-GCCCGGGC and cleaves double-stranded DNA after the third C in the sequence, producing blunt ends. SrfI is a rare-cutting enzyme and should therefore be useful for megabase mapping.

Epstein Barr virus/complement C3d receptor is an interferon alpha receptor.

Interferon alpha contains a sequence motif similar to the complement receptor type two (CR2/CD21) binding site on complement fragment C3d. Antibodies against a peptide with the CR2 binding sequence on C3d react with a peptide carrying the IFN alpha CR2 binding motif (residues 92-99) and with recombinant IFN alpha. The IFN alpha-derived peptide, as well as recombinant IFN alpha, inhibits C3bi/C3d interaction with CR2 on the Burkitt lymphoma Raji. The direct interaction of IFN alpha and CR2 is inhibited by polyclonal anti-IFN alpha, anti-CR2 and anti-C3d peptide antibodies as well as by C3bi/C3d, EBV coat protein gp350/220 and IFN but not by IFN gamma. [125I]IFN alpha binding to Raji cells is inhibited by polyclonal anti-IFN alpha and anti-CR2 antibodies, by peptides with the CR2 binding motif and partially by C3bi/C3d. Monoclonal anti-CR2 antibody HB5, but not OKB-7, blocks IFN alpha binding to Raji cells. CR2 or CR2-like molecules may therefore be the major IFN alpha receptors on B lymphocytes.

Fatty acid requirement of B lymphocytes activated in vitro.

The lipid and fatty acid requirement of B lymphocytes activated in vitro was examined by replacing soybean lipids with various combinations of lecithins, fatty acids and cholesterol. It is reported here that linoleic acid is the sole fatty acid required to support the proliferation of B lymphocytes and maturation to immunoglobulin-secreting cells.

Expression of oncogenes in normal and transformed murine B lymphocytes.

Proliferating, lipopolysaccharide-stimulated murine B lymphoblasts and a number of transformed murine B cell lines representing various differentiation stages of B cell lineage all express the myc, H-ras and K-ras oncogenes. N-ras transcripts are also present in the B cell blasts and some of the cell lines. In addition, abl, fms, fos, myb, src, and yes are transcribed in some or all of the cell lines but not in the normal B cell blasts. Only myb is expressed in a differentiation stage-specific manner; transcripts are present in the pre-B cell lines and a few of the B lymphomas but not in any plasmacytomas tested. The erbB, fes, mos, and sis probes do not hybridize with mRNA from any of the 15 cell lines or normal B cells. ErbA is not detectable in transformed cells but is expressed in the normal B cell blasts at a low level suggesting its possible involvement in normal growth regulation.

The role of C3 and its fragments in the control of S phase entry of activated mouse B lymphocytes via the complement receptor type 2.

Complement component C3 and receptors for its activated fragments have been recognized as regulators of humoral immune responses. Cross-linked C3b and C3d will substitute for alpha B cell growth factors (alpha BCGF) produced by macrophages by stimulating activated murine B lymphocytes to enter S phase. In contrast, soluble C3d and synthetic oligopeptides--containing the amino acid sequence of the binding site of C3d to its receptor, the complement receptor type 2--inhibit the entry into S phase of alpha BCGF-stimulated B cells. There is evidence supporting the hypothesis that a portion of the C3 gene at the 3' end, encoding parts of the alpha chain, encodes an alternate C3 product with alpha BCGF activity.

Three restriction points in the cell cycle of activated murine B lymphocytes.

The cell cycle of activated B lymphocytes was found to be controlled by three restriction points. The first occurs immediately after mitosis and was found to be controlled by the binding of Sepharose-bound, immunoglobulin-specific antibodies to surface membrane-bound immunoglobulin. Exposure to this stimulus as short as 15 min or as long as 36 hr allowed B cells to move into the G1 phase up to the next restriction point. The second restriction point was observed to be approximately equal to 4 hr after mitosis, in the G1 phase of the cycle and 3-4 hr before the B cells entered S phase, and was found to be controlled by alpha-type B-cell growth factors produced by the P388D1 macrophage line. A third restriction point occurs in the G2 phase, 2-4 hr before mitosis, and is apparently controlled by beta-type B-cell growth factors that are likely to be produced by helper T lymphocytes.

Activation and cell cycle control of murine B lymphocytes.

The cell cycle of activated murine B lymphocytes (B cells) is controlled by the occupancy of surface membrane-bound immunoglobulin (Ig) and by two types of growth factors, called alpha and beta factors. These growth factors are produced in an endocrine fashion by the interaction of helper T lymphocytes (T cells) with antigen-presenting macrophages (A cells). Antigen is taken up, processed and presented on the surface of A cells in the context of class II major histocompatibility complex (MHC) glycoproteins. Helper T cells recognize this association of antigen and class II MHC molecules. A cells produce alpha factors and T cells produce beta factors. The molecular nature of these factors and of the corresponding receptors on B cells has yet to be elucidated, although it can be shown that the complement component C3d replaces alpha factor action. Resting, G0 phase B cells are refractory to the action of alpha and beta factors. They have to be excited, i.e. rendered susceptible to the action of these factors. This can be achieved by the interaction with helper T cells that recognize antigen, bound by surface membrane Ig, in the context of class II MHC glycoproteins on the surface of resting G0 B cells. Excitation can also occur in a polyclonal fashion by cross-linking of surface Ig with immobilized, Ig-specific antibodies, or by the interaction with polyclonal activators of B cells, such as lipopolysaccharides. Entry into the cell cycle is asynchronous. Activated, cycling B cells can be synchronized by size separation, using velocity sedimentation. Synchronized B cells will retain their synchrony for several divisions, when they are stimulated by immobilized Ig-specific antibodies, alpha and beta factors. They divide every 20 h at 37 degrees C. Omission of either of the three stimuli arrests B cells, though at different points in the cell cycle. Three restriction points are found: the first occurs immediately after mitosis and is controlled by the binding of immobilized Ig-specific antibodies to surface membrane-bound Ig.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular interaction and the environment in lymphocyte development: the roles of antigen, histocompatibility, and growth factors in T cell-dependent B cell stimulation.

T cell-dependent B cell stimulation depends on cellular interactions between macrophages, T cells, and B cells. T cells recognize antigen in the context of Ia determinants on macrophages. This leads to the activation of helper T cells and to the production of helper factor(s) either by the activated helper T cells or by the macrophages. Cloned lines of helper T cells produce factors("help') for B cell replication and Ig secretion in the presence of histocompatible macrophages and of specific antigen. These factors stimulate histocompatible, as well as histoincompatible, mitogen-activated B cell blasts polyclonally. Thus, neither antigen nor histocompatibility, but antigen-unspecific, soluble factors are required to stimulate an activated B cell blast through the next round of division. Small resting B cells, however, are not stimulated to replication by these factors, but only to polyclonal, H-2-unrestricted maturation to Ig-secreting cells. Replication (and Ig secretion) of small resting B cells, however, is only induced when antigen-specific small B cells bind their specific antigen via surface Ig molecules and interact with histocompatible helper T cells. The preference of the resting B cells for such collaboration with T cell help is mapped to the K-end of the H-2 locus and probably constitutes the antigen expressed on B cells by the I region. It appears that a resting B cell is excited by the binding of specific antigen to surface Ig and by the interaction of its surface Ia with helper T cells. After this dual recognition the excited B cell can be stimulated by the antigen-unspecific factor(s) generated by the interaction of helper T cells, adherent cells and antigen to initiate growth. Immune induction of a B cell thus involves three controlling elements: Ig, Ia, and B cell growth factor receptors.

Antigen-specific T-helper cells stimulate H-2-compatible and H-2-incompatible B-cell blasts polyclonally.

Lipopolysaccharide (LPS)-activated B-cell blasts from C57BL/6J nu/nu spleen cells develop into IgM-secreting clones after stimulation by antigen-specific T-helper cells of C57BL/6J origin. Although induction of help is antigen-dependent, help itself acts polyclonally. 1 of 1--3 B-cell blasts is restimulated in a homologous fashion by LPS, or in a heterologous fashion by sheep erythrocyte (SRC)- or horse erythrocyte (HRC)-activated T-helper cells. The repertoire of activated B-cell blasts reflects the polyclonal nature of activation: approximately 1 in 1,000--3,000 restimulated B-cell blasts is specific for SRC, 1 in 300--1,000 is specific for HRC, and 1 in 100--300 specific for trinitrophenylated SRC (TNP30-SRC). B-cell blasts that are either H-2 compatible or H-2 incompatible with the antigen-activated T-cell help are stimulated polyclonally in similar high frequencies. Thus, neither antigen nor H-2 compatibility are required to stimulate a B-cell blast into the next cell cycle.

The purified protein derivative of turberculin, a B-cell mitogen that distinguishes in its action resting, small B cells from activated B-cell blasts.

The purified protein derivative of tuberculin (PPD tuberculin) stimulates approximately one of two lipopolysaccharide (LPS)-activated B-cell blasts of C57BL/6J nu/nu spleen cells to continued clonal growth and maturation to IgM and IgG secretion. It alwo stimulates background, in vivo-activated large cells of normal C57BL/6J nu/nu spleen to growth and Ig secretion, at a frequency of approximately 1 of 100 large spleen cells. PPD tuberculin, therefore, is a polyclonal B-cell activator for B-cell blasts. Many single murine splenic B cells (approximately 50%) appear to have reactivities, and therefore probably receptors, for LPS and PPD tuberculin. PPD tuberculin does not stimulate small, resting B cells to growth as measured by the number of cells in culture and by thymidine uptake. However, it stimulates approximately one-fourth of all spleen cells to blast transformation. The large-size blast cells secrete IgM and, therefore, form plaques in the protein A plaque assay. IgG-secreting, plaque-forming cells develop at later stages of stimulation, indicating that the switch from IgM to IgG may occur without division in single, stimulated B cells. Stimulation of resting B cells to maturation by PPD tuberculin is polyclonal. Thus, approximately 1 in 10(2) IgM-secreting plaque-forming cells form plaques with trinitrophenyl-substituted sheep erythrocytes, 1 in 450 do so with horse erythrocytes, and 1 in 10(3) with sheep erythrocytes. Furthermore, the number of Ig-secreting cells developing from small, resting cells without growth in cultures with or without filler thymus cells suggests polyclonal activation by PPD tuberculin to maturation only of at least one out of four small, splenic B cells.