The HysNiche trial: hysteroscopic resection of uterine caesarean scar defect (niche) in patients with abnormal bleeding, a randomised controlled trial.

BACKGROUND: A caesarean section (CS) can cause a defect or disruption of the myometrium at the site of the uterine scar, called a niche. In recent years, an association between a niche and postmenstrual spotting after a CS has been demonstrated. Hysteroscopic resection of these niches is thought to reduce spotting and menstrual pain. However, there are no randomised trials assessing the effectiveness of a hysteroscopic niche resection. METHODS/DESIGN: We planned a multicentre randomised trial comparing hysteroscopic niche resection to no intervention. We study women with postmenstrual spotting after a CS and a niche with a residual myometrium of at least 3 mm during sonohysterography. After informed consent is obtained, eligible women will be randomly allocated to hysteroscopic resection of the niche or expectant management for 6 months. The primary outcome is the number of days with postmenstrual spotting during one menstrual cycle 6 months after randomisation. Secondary outcomes are menstrual characteristics, menstruation related pain and experienced discomfort due to spotting or menstrual pain, quality of life, patient satisfaction, sexual function, urological symptoms, medical consultations, medication use, complications, lost productivity and medical costs. Measurements will be performed at baseline and at 3 and 6 months after randomisation. A cost-effectiveness analysis will be performed from a societal perspective at 6 months after randomisation. DISCUSSION: This trial will provide insight in the (cost)effectiveness of hysteroscopic resection of a niche versus expectant management in women who have postmenstrual spotting and a niche with sufficient residual myometrium to perform a hysteroscopic niche resection. TRIAL REGISTRATION: Dutch Trial Register NTR3269 . Registered 1 February 2012. ZonMw Grant number 80-82305-97-12030.

Prevalence, potential risk factors for development and symptoms related to the presence of uterine niches following Cesarean section: systematic review.

OBJECTIVE: To review systematically the medical literature reporting on the prevalence of a niche at the site of a Cesarean section (CS) scar using various diagnostic methods, on potential risk factors for the development of a niche and on niche-related gynecological symptoms in non-pregnant women. METHODS: The PubMed and EMBASE databases were searched. All types of clinical study reporting on the prevalence, risk factors and/or symptoms of a niche in non-pregnant women with a history of CS were included, apart from case reports and case series. RESULTS: Twenty-one papers were selected for inclusion in the review. A wide range in the prevalence of a niche was found. Using contrast-enhanced sonohysterography in a random population of women with a history of CS, the prevalence was found to vary between 56% and 84%. Nine studies reported on risk factors and each study evaluated different factors, which made it difficult to compare studies. Risk factors could be classified into four categories: those related to closure technique, to development of the lower uterine segment or location of the incision or to wound healing, and miscellaneous factors. Probable risk factors are single-layer myometrium closure, multiple CSs and uterine retroflexion. Six out of eight studies that evaluated niche-related symptoms described an association between the presence of a niche and postmenstrual spotting. CONCLUSIONS: The reported prevalence of a niche in non-pregnant women varies depending on the method of detection, the criteria used to define a niche and the study population. Potential risk factors can be categorized into four main categories, which may be useful for future research and meta-analyses. The predominant symptom associated with a niche is postmenstrual spotting.

Clustering of dendritic cells, helper T lymphocytes, and histocompatible B cells during primary antibody responses in vitro.

Mouse spleen suspensions generate discrete cell clusters within 1-2 d of culture. We have isolated these clusters by velocity sedimentation to study their contribution to primary antibody responses. Clusters represent approximately 5% of the starting spleen cells and consist of 20-50% B cells, 20-50% T cells, and 10-20% dendritic cells (DC). When the cultures are stimulated with thymus-dependent antigens, like heterologous red cells or dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH), the clusters are the principal site for the development of plaque-forming cells (PFC). Noncluster fractions form few PFC and only when supplemented with fresh DC. PFC responses in all cases are antigen specific. B cells cluster only in the presence of T cells and DC (1 DC/200 B-T cell mixtures) and only after encountering specific antigen. The elimination of either DC or Lyt-1+2- T cells, with monoclonal antibody and complement, ablates B cell development into PFC. PFC responses are restored with antigen-nonspecific helper factors formed in the syngeneic mixed leukocyte reaction between DC and T cells. Since PFC to DNP-KLH do not develop de novo when B cells are exposed to antigen and helper factors, anti-DNP PFC precursors must be stimulated within clusters to become responsive to helper factors. PFC development within clusters is restricted by the major histocompatibility complex (MHC). When DC and T cells are from strain P1, then P1 but not P2 B cells develop into PFC; when DC are from strain P2 and T cells from strain P1, strain P2 B cells are selected to become PFC in clusters. The entry of B cells into clusters is itself MHC restricted, since P1 DC/T cells aggregate six times as many B cells from strain P1 as strain P2. Thus, clusters are the site in which DC, B, and T cells interact to generate PFC. One can use clusters to retrieve B cells that have been selected in an antigen-dependent, MHC-restricted fashion and to show that clustering B cells become responsive to soluble, polyclonal helper factors.

Dendritic cells are the principal stimulators of the primary mixed leukocyte reaction in mice.

Clone 33D1 is a mouse-rat hybridoma that secretes a specific anti-dendritic cell (DC) monoclonal antibody (14). Because the antibody kills DC in the presence of rabbit complement, it can be used to study the functional consequences of selective DC depletion. Previous data on the cell specificity of 33D1 were first extended. By cytotoxicity (rabbit complement) and indirect immunofluorescence (biotin-avidin technique), 33D1 reacted with DC but not with macrophages nor other splenocytes. In contrast, the monoclonal antibody, F4/80 (15), reacted with macrophages but not DC. The functional assay evaluated in this paper was stimulation of the primary mixed leukocyte reaction (MLR). 33D1 antibody itself did not inhibit stimulation by enriched populations of DC. In the presence of complement, 33D1 killed DC and ablated stimulatory function. The effect of 33D1 and complement on MLR stimulation by heterogenous cell mixtures was then evaluated. Removal of DC from unfractionated spleen suspensions reduced stimulatory capacity 75-90 percent, comparable to that produced with specific anti-Ia antibody and complement. Stimulation of both proliferative and cytotoxic responses was reduced. DC depletion had similar effects on MLR generated across full strain differences, or across selected subregions (H2I, H-2K/D) of the major histocompatibility complex. To further compare the functional properties of spleen DC and macrophages, MLR stimulation by adherent and nonadherent fractions of spleen were tested separately. 62 +/- 8 percent of the total stimulatory capacity of spleen was in the plastic adherent population. Activity was ablated greater than 90 percent after elimination of DC. MLR stimulation by 24-h cultures of spleen adherent cells, which contained a three- to sixfold excess of Ia(+) macrophages, was also ablated when DC were removed. Stimulation by nonadherent spleen was more resistant, but was reduced 50-75 percent by 33D1 and complement. The function of spleen cells treated with 33D1 or anti-Ia antibody and complement was restored with a small inoculum of purified DC. The latter corresponded to 0.5 percent of total stimulator cells and were enriched by previously described techniques that did not require the 33D1 antibody. We conclude that the DC, a trace component of mouse spleen, is the principal cell type required for stimulation of the primary MLR. Because other cells are not immunogenic, but do express Ia and H-2 alloantigens, DC likely represent the critical accessory cell required for the induction of lymphocyte responses.

Identification of a novel cell type in peripheral lymphoid organs of mice. V. Purification of spleen dendritic cells, new surface markers, and maintenance in vitro.

Dendritic cells (DCs; 1) have been purified from mouse spleen in good yield. Spleen cell suspensions were floated on dense bovine plasma albumin (BPA) columns, and the low density fraction was adhered to glass (2). The adherent cells consisted of DCs and immature macrophages most of which eluted in a viable state from the culture dish after overnight incubation. The macrophages were then removed by selective rosetting with opsonized erythrocytes and recentrifugation on dense BPA. This protocol resulted in a purified DC fraction, containing 1--3 X 10(5) DCs/spleen, which was homogeneous and distinctive in its properties. All cells exhibited the phase contrast and transmission electron microscopy (EM) cytologic features that were previously described for freshly isolated adherent DCs. By scanning EM, most purified DCs exhibited a remarkable array of bulbous protrusions of varying length and shape, unlike any other lymphoid cell. All DCs expressed surface Ia and other major histocompatibility complex (MHC)-linked alloantigens. DCs, however, lacked surface Ig and T-cell antigens, and did not bind or interiorize opsonized erythrocytes. Purified DCs have been maintined in vitro for 3 days. Recovery of cultured purified cells was 70% or more of starting cell numbers. When [3H]uridine-tagged DCs were mixed with nonlabeled heterogeneous spleen cells, 70--80% of the labeled DCs were recovered as viable cells 2--3 days later. Purified DCs did not readhere to tissue culture surfaces and did not proliferate, even when cultured with mitogenic doses of concanavalin A and lipopolysaccharide. Finally, DCs did not change their cytologic or surface properties after 3 days of culture. These observations extend the evidence that DCs are a novel cell type and provide useful properties and techniques for their further study.

Lymphokine enhances the expression and synthesis of Ia antigens on cultured mouse peritoneal macrophages.

Soluble products from antigen stimulated Trypanosoma cruzi-immune spleen cells enhanced the expression of Ia antigens on proteose-peptone-elicited mouse peritoneal macrophages (M phi). Acquisition of Ia paralleled M phi activation, previously shown to be mediated by this same source of lymphokine (LK). Expression of Ia and four other plasma membrane antigens was monitored by quantitative binding and radioautographic studies with 125I-monoclonal antibodies. Immune LK selectively enhanced expression of Ia and, to a lesser extent, H-2D relative to control LK from antigen-stimulated noninfected spleen. The levels of three other non-major histocompatibility complex (MHC) antigens, including the trypsin-resistant Fc receptor, were similar in cells exposed to both sources of LK. As little as 1% immune LK induced one-half maximal expression of Ia. Kinetic studies revealed that much of the Ia on freshly explanted peritoneal M phi was lost during the 1st d of culture. In the continued presence of immune LK, Ia was re-expressed on virtually all M phi by the 2nd and 3rd d. Alternatively, > 95% Ia negative populations were obtained by culturing the cells 3 d; then, addition of LK induced Ia on most cells within 1 d. Once induced, Ia persisted on the M phi surface for at least 2 d. [35S]methionine radiolabeling indicated that immune LK selectively increased radiolabeling of M phi Ia, again with other non-MHC-linked plasma membrane polypeptides as controls. LK-induced Ia-bearing M phi were tested as primary mixed leukocyte reaction stimulators. 1 x 10(5)-2 x 10(5) M phi did not stimulate 4.5 x 10(6) responding T cells, whereas 10(4) dendritic cells induced strong responses, as previously described. Because Ia-positive M phi do not actively sensitize T cells in a model immune response, we propose that M phi MHC products serve primarily as recognition sites for previously sensitized T cells, thereby enhancing T cell-mediated M phi activation.

Immunologic properties of purified epidermal Langerhans cells. Distinct requirements for stimulation of unprimed and sensitized T lymphocytes.

Langerhans cells (LC) are Ia+ leukocytes that account for less than 2% of the cells in murine epidermal isolates. We purified LC by cell sorting to study their capacity to stimulate antigen-specific responses from unprimed and sensitized T cells. Sorting was performed after 12 or 72 h of epidermal culture, since our earlier work had indicated that LC became immunologically active during that time interval. At 12 and 72 h, the LC were uniformly and equally rich in the Ia glycoproteins that are recognized by helper T cells. At both time points, LC were comparable in their capacity to stimulate sensitized helper T lymphocytes, and would cluster the T cells in an antigen-dependent fashion at 4 degrees C. However, 12-h LC did not sensitize T cells, as indicated by their inactivity in stimulating the primary MLR or antibody response, and they were unable to cluster T cells in an antigen-independent fashion at 37 degrees C. The latter properties were acquired during 72 h of culture. As a result, the function of 72-h LC fully resembled that of lymphoid dendritic cells. We propose that the maturation of stimulatory function within the dendritic cell lineage represents an important control point in the induction phase of cell-mediated immunity.

Distribution and turnover of Langerhans cells during delayed immune responses in human skin.

The changes in distribution and turnover of T6+ Langerhans cells (LC) in the skin during delayed immune responses to tuberculin, and in the lesions of tuberculoid leprosy and cutaneous Leishmaniasis were investigated. In each situation, there was a dermal accumulation of monocytes and T cells and epidermal thickening with keratinocyte Ia expression. In the tuberculin response a dramatic change in the distribution of LC was observed. By 41 h, T6+ LC were displaced to the upper zone of the thickening epidermis followed by an almost complete loss of LC from the epidermis by approximately 72 h. After 7 d, T6+ cells started to reappear in the epidermis, which regained almost normal numbers of T6+ LC by 14 d. After antigen administration and initiation of the delayed immune response, enhanced numbers of T6+ cells appeared in association with the mononuclear cell infiltrate of the upper dermal lesions. Their numbers peaked by 72 h, were reduced at 7 d, and again enhanced by 14 d, when the epidermis was being repopulated. Similar numbers of T6+ cells were found in the chronic lesions of tuberculoid leprosy and cutaneous Leishmaniasis but not lepromatous leprosy. The cells of the dermis were identified as typical LC by the presence of Birbeck granules and surface T6 antigen at the electron microscope level. These cells were closely associated with lymphocytes. We have quantified the number of LC, evaluated their directional flux into the epidermis and dermis, determined nearest neighbors, and made predictions as to their fate.

Specific antimononuclear phagocyte monoclonal antibodies. Application to the purification of dendritic cells and the tissue localization of macrophages.

3C10 and 1D9 are two related monoclonal antibodies that specifically identify human mononuclear phagocytes in a large number of sites, including blood monocytes, alveolar macrophages, and macrophages in tissue sections of spleen, lymph node, and skin. The antigen persists on monocytes cultured for greater than 4 wk, but it is not found on giant cells. The 3C10-1D9 determinant is carried by a 55 kD polypeptide, is expressed at approximately 40,000 copies per monocyte, and is protease sensitive. The antigen is clearly different from HLA-class II or Ia-like antigens that have been studied with a new monoclonal 9.3F10. The 9.3F10 antigen is found on B cells, dendritic cells and monocytes; is protease resistant, and occurs on a 33-29 kD doublet typical of class II products. The 3C10 monoclonal provides a clear distinction between human mononuclear phagocytes and dendritic cells. First, monocytes and lymphocytes can be eliminated from plastic-adherent mononuclear cells using 3C10, complement, and two previously described cytotoxic antibodies, BA-1 (anti-B cell) and Leu-1 (anti-T cell). As a result, the trace dendritic cell component of blood can be enriched to considerable purity (65-75%) and yield. Second, immunocytochemical staining of tissue sections reveals that 3C10+ macrophages are anatomically segregated from dendritic cells. Large numbers of 3C10+ cells are found in red pulp of spleen and in regions surrounding lymphatic channels of lymph node. However, 3C10+ macrophages are scarce in white pulp of spleen and the lymphocyte-rich cortex of node that are the sites where dendritic cells are localized. 3C10+ cells in skin are found in the dermis, particularly in leprosy infiltrates, but the Langerhans' cells of epidermis are 3C10-. The distinctive localization of macrophages and dendritic cells is consistent with their respective functions as effector and accessory cells in the immune response.

Blockade of clearance of immune complexes by an anti-Fc gamma receptor monoclonal antibody.

Clearance of immune complexes by the mononuclear phagocyte system is important for maintaining normal host defenses against bacterial and viral assault (1), but also contributes to the pathogenesis of a variety of immune- mediated diseases . For example, removal from the circulation of IgG-coated erythrocytes and platelets by the MPS is the sine qua non of immune-mediated cytopenias (2, 3). On the other hand, abnormally decreased removal by the MPS of smaller, soluble immune complexes may play a role in the pathogenesis of immune complex-mediated tissue damage found in such autoimmune diseases as SLE (4). Although the physicochemical nature and the size of immune complexes can influence rates of clearance and sites of deposition (reviewed in 5), interactions between immune complexes and the MPS in vivo are poorly understood. The inability to directly measure binding or internalization of immune complexes by cells in the liver and spleen has made the analysis of the molecular basis of immune complex clearance very difficult . Receptors for the Fc portion of IgG (FcgammaR) and for complement (CR) undoubtedly play a role in the removal of immune complexes, but the relative importance of these receptors is not known.

Studies of the cell surface of mouse dendritic cells and other leukocytes.

The surface of dendritic cells (DC) has been analyzed by means of monoclonal antibodies (Ab) and lactoperoxidase (LPO)-mediated radioiodination. Antigens and other exteriorily disposed polypeptides of purified spleen DC were compared with those of tissue macrophages (Mphi), monocytes, and other bone marrow-derived elements. Quantitative binding studies and autoradiography with (125)I-Ab established that DC expressed high levels of I-A and H-2D, 2 x 10(5) and 1 x 10(5) Ab binding sites per cell, respectively. DC from conventional, germ-free, and specific pathogen-free mice were all rich in Ia. Expression of Ia on B cells was 5-10 percent of that on DC and increased fivefold during lipopolysaccharide mitogenesis. More than 70-90 percent of purified Mphi and monocytes from specific pathogen-free mice were Ia negative, but increased levels of Ia were noted on cells from mice reared under conventional conditions. Thus large amounts of Ia on DC is a constitutive trait, whereas the expression of Ia by other cell types may be governed by the environmental and immunological status of the host. The 2.4G2 Fc receptor Ag was not detected on DC. Peritoneal and spleen Mphi had 10(5) 2.4G2 binding sites/cell, whereas monocytes and lymphocytes were less reactive (1 x 10(4)-3 x 10(4) binding sites/cell). Four other Mphi-related antigens were evaluated. Each had a distinctive tissue distribution and none bound exclusively to Mphi and monocytes. Neither 1.21J (Mac-1) nor F4/80 reacted with DC. Immunoprecipitation studies of externally ((125)I) and biosynthetically ([(35)S]methionine)dabeled cells confirmed the binding data. Sensitive binding assays with (125)I-Ab confirmed previous observations that DC lack Ig and Thy-1. Lyt-1 was also not found on DC, but 5-12 percent of the cells in purified DC preparations expressed both Lyt-2 and Ia. All DC expressed the leukocyte common antigens at levels similar to other leukocytes. The spectrum of surface polypeptides labeled by LPO-mediated iodination was different on Mphi, DC, and lymphocytes. Polypeptides migrating at molecular weights of 155,000, 85,000, and 62,000 appeared to be restricted to DC. These observations establish that the cell surface of DC differs considerably from other leukocytes, including the blood monocyte, and suggest that the DC is part of a unique Ia-rich leukocyte differentiation pathway.

The Thy-1-bearing cell of murine epidermis. A distinctive leukocyte perhaps related to natural killer cells.

Bone marrow-derived leukocytes of murine epidermis can express two phenotypes: typical Langerhans cells, which are Ia+ and Thy-1-, and a recently discovered second population that is Thy-1+ and Ia-. To verify that these phenotypes are expressed by two different cell types, and to help understand their lineage and function, we have studied morphology and reactivity with a large panel of antibodies. Dual antibody immunofluorescence combined with electron microscopy showed that Thy-1+ and Ia+ cells were each distributed in a regular fashion and formed adjacent dendritic systems in or close to the basal layer. Double-labeling studies with anti-Ia and a second monoclonal antibody revealed that all Langerhans cells expressed F4/80 (macrophage), Mac-1 (C3bi receptor), and 2.4G2 (Fc receptor), as well as the thymus leukemia (TL) and heat-stable (M1.69/16) antigens. A large fraction expressed S100 and all exhibited membrane ATPase and nonspecific esterase. In contrast, Thy-1+ cells lacked all these features of Langerhans cells, except that a minority were strongly reactive with 2.4G2. Thy-1+ cells also lacked differentiation antigens of most other types of leukocytes, except they were rich in asialo GM1. By electron microscopy, Thy-1+ cells had cytoplasmic granules that were similar in structure and in their aryl sulfatase content to those previously described in natural killer cells. The granules were enlarged in beige mice, suggesting a lysosomal origin, and were present in mast cell-deficient W/Wv mice, indicating no relation to mast cells. We conclude that Thy-1+ epidermal cells are thoroughly distinct from Langerhans cells. On the basis of morphology and phenotype, they may represent a type of tissue natural killer cell. Thy-1+ natural killer cells are now being identified in several nonlymphoid sites, such as gut epithelium and the livers of mice given adjuvants. If Thy-1+ epidermal cells prove to be natural killer cells, it is noteworthy that they represent a resident population regularly distributed in the basal layer of all mouse strains. The notion that Thy-1+ epidermal cells are immature natural killer cells is intriguing in light of recent evidence that Ia+ Langerhans cells are also immature with respect to accessory cell function. The epidermis may not have the functional capacities of a lymphoid organ, but it could contribute immature cells important for both natural and acquired resistance.

Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells.

Integrase is essential for human immunodeficiency virus-type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.

X-ray structure of simian immunodeficiency virus integrase containing the core and C-terminal domain (residues 50-293)--an initial glance of the viral DNA binding platform.

The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.

Probing the catalytic roles of n2-site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis.

The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn(2+)-KD values, UV-cis and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn(2+)-activated enzymes showed reduced kcat/Km values for ATP (> 7-fold) and for glutamate (> 10-fold). Of the unadenylylated Mg(2+)-activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20-fold better catalyst than WTGS. We propose the n2-site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate gamma-glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition-state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady-state kinetic data suggest that modifying the charge density increases the transition-state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X-ray structures of GS.

2-Aryl-2,2-difluoroacetamide FKBP12 ligands: synthesis and X-ray structural studies.

[structure: see text] 2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose rotamase inhibitory activity is comparable to that seen for the corresponding ketoamides. X-ray structural studies suggest that the fluorine atoms participate in discrete interactions with the Phe36 phenyl ring and the Tyr26 hydroxyl group, with the latter resembling a moderate-to-weak hydrogen bond.

The sensitization phase of T-cell-mediated immunity.

Many cell-mediated immune responses appear to develop in two phases: A sensitization phase in which unprimed or memory T cells interact with dendritic cells to become active lymphoblasts, and an effector phase in which the T lymphoblasts and other presenting cells interact to eliminate the antigen. Antigen presentation is essential to both phases. Here we review several features that are pertinent to the special sensitization role of dendritic cells. First, dendritic cells from lymphoid tissues, blood, and lymph (lymphoid dendritic cells) express very high levels of class I and II MHC products, and these levels cannot be increased by exposure to cytokines such as immune interferon. Second, dendritic cells efficiently cluster antigen-specific T cells during primary responses. Other presenting cells, like macrophages and B lymphocytes, do not form clusters but do bind to sensitized T lymphoblasts. Dendritic-T-cell binding is not inhibited by mAb to CD4 and LFA-1 antigens. It is suggested that a dendritic-cell-specific molecule is required. Third, it is not yet clear if dendritic cells make a "lymphocyte activating factor." However, IL-1 is not produced, even when dendritic cells are in contact with responding T cells. Fourth, dendritic cells have the capacity to migrate from the tissues and move to T-dependent areas. Epidermal Langerhans cells represent a reservoir of tissue dendritic cells but seem to be immunologically immature. The viability and accessory function of the Langerhans cell greatly depend on a single cytokine, granulocyte-macrophage colony stimulating factor (GM-CSF), leading to the proposal that GM-CSF is critical in mobilizing active dendritic cells at the onset of a cell-mediated immune response.

Isolation of Chinese hamster ovary cells with reduced poly(ADP-ribose) polymerase activity.

The biological function of poly(ADP-ribose) polymerase in DNA repair, cell-cycle regulation and cellular differentiation has yet to be defined. Isolation of cells which are deficient in poly(ADP-ribose) synthesis would greatly facilitate the determination of the biological role of this enzyme. A method is described for isolating Chinese hamster ovary (CHO) cells deficient in the poly(ADP-ribose) polymerase activity by direct screening of colonies for enzyme activity. Colonies with decreased production of poly(ADP-ribose) are recovered from nylon replicas for further analysis. Using this method we have isolated a series of CHO cells which have 50% or less poly(ADP-ribose) polymerase activity. These mutants have normal generation times and are 20% more sensitive to the effects of DNA (m)ethylating agents than the parental cell. However, these mutants display normal sensitivity to gamma-rays.

Increased sensitivity to DNA-alkylating agents in CHO mutants with decreased poly(ADP-ribose) polymerase activity.

Using a replica-plating procedure and a 32P-NAD+ permeable cell-screening assay, we have isolated a CHO mutant, PADR-9, which displays approximately 17% of the wild-type level of poly(ADP-ribose) polymerase activity. Biochemical analysis of the mutant using activity, Western, and Northern blot techniques indicate that relative to its parent cell, the mutant's enzyme activity, antibody recognition, and mRNA levels have been reduced to approximately the same extent. These results are consistent with a mutation in the PADR-9 cell which has resulted in a reduction in enzyme synthesis due to reduced mRNA synthesis and/or stability. Relative to wild-type CHO cells, the PADR-9 mutant has increased sensitivity to killing by DNA-alkylating agents but has normal gamma-ray sensitivity. Correlation between a decrease in poly(ADP-ribose) polymerase activity and an increased sensitivity to DNA-alkylating agents suggests that poly(ADP-ribose) synthesis may be important in the repair and/or induction of DNA damage produced by these agents.