Survey of current practices and opinions of German Society of Gynecologic Endoscopy members regarding the treatment of ovarian neoplasia by robotic surgery.

PURPOSE: As data on this topic are sparse and contradictory, we aimed to ascertain the opinions of the members of the German Society of Gynecologic Endoscopy (AGE) regarding the use of robotic surgery in the treatment of ovarian malignancies. METHODS: In 2015, an anonymous questionnaire was sent to AGE members to assess their views on the treatment of ovarian malignancies by robotic surgery according to T stage and the current treatment practices in their facilities. RESULTS: Of the 228 respondents, 132 (58%) were fellows or attending physicians and 156 (68%) worked at university hospitals or tertiary referral centers. Most [n = 218 (96%)] respondents reported treating < 10% of their patients using robotic surgery. Respondents felt that T1 and borderline ovarian tumors, but not T2 (51%) or T3/4 (76%) tumors, should and could be treated by robot surgery. 162 (71%) respondents considered the currently available data on this subject to be insufficient, and 42% indicated their willingness to participate in clinical studies on the applicability of robotic surgery to the treatment of T1/2 ovarian tumors. CONCLUSION: The majority of AGE members surveyed considered robotic surgery to be an option for the treatment of T1 ovarian malignancies and borderline ovarian tumors. However, prospective randomized studies are needed to determine the relevance of robotic surgery in this context.

Impact of different intraoperative CO2 pressure levels (8 and 15 mmHg) during laparoscopic hysterectomy performed due to benign uterine pathologies on postoperative pain and arterial pCO2 : a prospective randomised controlled clinical trial.

OBJECTIVE: To compare the effects of two different intraoperative CO2 pressures (8 and 15 mmHg) during laparoscopic hysterectomy for benign uterine pathologies in terms of postoperative abdominal and shoulder pain, laparoscopy-mediated vegetative alterations, pain medication requirement, arterial CO2 pressure (pCO2 ), surgical parameters, and safety. DESIGN: Prospective randomised controlled study. SETTING: German university hospital. POPULATION: Female patients undergoing laparoscopic hysterectomy for benign uterine pathologies. METHODS: Patients were randomised to a standard pressure (SP; 15 mmHg, control) or low-pressure (LP; 8 mmHg, experimental) group. MAIN OUTCOME MEASURES: Primary outcomes were postoperative abdominal and shoulder pain intensities, measured via numeric rating scale (NRS) and vegetative parameters (fatigue, nausea, vomiting, bloating) at 3, 24, and 48 hours postoperatively. Secondary outcomes were pain medication requirement (mg) and arterial pCO2 (mmHg). Surgical parameters and intra- and postoperative complications were also recorded. RESULTS: In total, 178 patients were included. Patients in the LP group (n = 91) showed significantly lower postoperative abdominal and shoulder pain scores, fewer vegetative alterations, lower pain medication requirements, a shorter postoperative hospitalization, and lower intra- and postoperative arterial pCO2 values compared with the SP group (n = 87; P ≤ 0.01). No differences in intra- and postoperative complications were observed between groups. CONCLUSIONS: Low-pressure laparoscopy seems to be an effective and safe technique for the reduction of postoperative pain and laparoscopy-induced metabolic and vegetative alterations following laparoscopic hysterectomy for benign indications. TWEETABLE ABSTRACT: Low-pressure laparoscopy seems to be an effective and safe technique for reduction of pain following laparoscopic hysterectomy.

Lymphadenopathy, elevated serum IgE levels, autoimmunity, and mast cell accumulation in flaky skin mutant mice.

The autosomal recessive mutation "flaky skin" (fsn) causes pleiotropic abnormalities in the immune and hematopoietic systems accompanied by pathologic changes in the skin. Homozygotes (fsn/fsn) showed increased size and histological alterations in the spleen and lymph nodes. Abnormalities in lymphoid architecture of the spleen in fsn/fsn mice were accompanied by marked increases in total numbers of B cells, macrophages, and immature erythroid cells. Splenic B cells displayed elevated MHC class II expression. Serum IgE levels were greater than 100 microg/ml by 10 weeks of age, representing > 7000-fold increase compared with normal littermates. This increased IgE level was associated with elevated IL-4 production by spleen cells and with increased amounts of serum IL-4. Serum IgM, IgG1, and IgG2b levels were also increased in fsn/fsn mice while IgG3 was decreased. Autoimmunity in fsn/fsn mice was evidenced by glomerulonephritis accompanied by immune complex deposition in the kidneys, increased serum blood urea nitrogen levels, and the presence of circulating anti-double-stranded DNA autoantibodies. Pathological changes in the skin of fsn/fsn mice were characterized by epidermal hyperplasia and mixed dermal inflammation. Increased numbers of mast cells were also observed in the dermis of the truncal skin as well as in the epithelial stomach. These marked immunological abnormalities suggest that the fsn locus encodes a major immunoregulatory molecule important in multiple immune and hematopoietic functions.

Increased G-CSF responsiveness of bone marrow cells from hematopoietic cell phosphatase deficient viable motheaten mice.

The mouse mutation viable motheaten (me(v)) results in defects in the expression and catalytic activity of the cytoplasmic protein tyrosine phosphatase known as hematopoietic cell phosphatase (HCP). This reduction in HCP activity leads to the aberrant regulation of several myeloid and lymphoid cell lineages, including substantial increases in numbers of granulocytes. The differentiation, proliferation, and survival of cells in this lineage are normally supported by granulocyte-colony stimulating factor (G-CSF). In this study we have determined the consequences of the loss of HCP activity in me(v)/me(v) mice on the response of bone marrow cells to G-CSF. Bone marrow from these mice exhibited substantial increases in clonogenic and proliferative responses to G-CSF. These enhanced activities of G-CSF correlated with an increase in the level of immature granulocytic, G-CSF receptor positive cells in the bone marrow. These results suggested the possibility that HCP may regulate the G-CSF receptor by a direct interaction. However, under conditions where the previously described interaction between the erythropoietin receptor and HCP was readily observed, HCP did not detectably associate with the G-CSF receptor.

Role of natural killer cells on engraftment of human lymphoid cells and on metastasis of human T-lymphoblastoid leukemia cells in C57BL/6J-scid mice and in C57BL/6J-scid bg mice.

The severe combined immunodeficiency (scid) mutation was backcrossed onto the C57BL/6J strain background in order to study the role of natural killer (NK) cells in rejection of normal and malignant human lymphohematopoietic cells. C57BL/6J-scid/scid mice showed severe loss of mature T and B cells accompanied by increased percentages of NK1.1+ cells and myeloid cells. Although little or no serum immunoglobulin was detectable prior to 2 months of age, all mice tested had circulating immunoglobulin by 7.5 months of age. C57BL/6J-scid/scid mice had markedly elevated levels of both hemolytic complement activity and NK cell activity compared with C57BL/6J - (+/+) controls. Weekly injections with anti-NK1.1 antibody resulted in elimination of NK cell activity in C57BL/6J-scid/scid mice throughout 8 weeks of treatment. Although human CEM-C7 T lymphoblastoid tumor cells grew slowly in unmanipulated C57BL/6J-scid/scid mice, anti-NK1.1 treatment resulted in increased growth accompanied by metastasis of human lymphoma cells to the brain, liver, and kidney. In contrast to T lymphoblastoid tumor cells, nonmalignant human peripheral blood mononuclear cells engrafted at low levels in anti-NK1.1-treated as well as in unmanipulated C57BL/6-scid/scid mice. Backcrossing of the beige (bgJ) mutation onto the C57BL/6-scid/scid genetic stock caused decreased NK cell activity accompanied by granulocyte defects. C57BL/6-scid/scid bgJ)/bgJ) mice showed metastasis of human CEM-C7 cells to the brain and other organs but supported only low levels of engraftment with human peripheral blood mononuclear cells. These results demonstrate that NK cells, in the absence of an adaptive immune system, function in resistance to metastasis of human lymphomas and suggest that innate immune factors in addition to NK cell function mediate resistance to engraftment of normal human peripheral blood leukocytes.

Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.

The scid mutation was backcrossed ten generations onto the NOD/Lt strain background, resulting in an immunodeficient stock (NOD/LtSz-scid/scid) with multiple defects in adaptive as well as nonadaptive immunologic function. NOD/LtSz-scid/scid mice lack functional lymphoid cells and show little or no serum Ig with age. Although NOD/(Lt-)+/+ mice develop T cell-mediated autoimmune, insulin-dependent diabetes mellitus, NOD/LtSz-scid/scid mice are both insulitis- and diabetes-free throughout life. However, because of a high incidence of thymic lymphomas, the mean lifespan of this congenic stock is only 8.5 mo under specific pathogen-free conditions. After i.v. injection of human CEM T-lymphoblastoid cells, splenic engraftment of these cells was fourfold greater in NOD/LtSz-scid/scid mice than in C.B17/Sz-scid/scid mice. Although C.B-17Sz-scid/scid mice exhibit robust NK cell activity, this activity is markedly reduced in both NOD/(Lt-)+/+ and NOD/LtSz-scid/scid mice. Presence of a functionally less mature macrophage population in NOD/LtSz-scid/scid vs C.B-17Sz-scid/scid mice is indicated by persistence in the former of the NOD/Lt strain-specific defect in LPS-stimulated IL-1 secretion by marrow-derived macrophages. Although C.B-17Sz-scid/scid and C57BL/6Sz-scid/scid mice have elevated serum hemolytic complement activity compared with their respective +/+ controls, both NOD/(LtSz-)+/+ and NOD/LtSz-scid/scid mice lack this activity. Age-dependent increases in serum Ig levels (> 1 micrograms/ml) were observed in only 2 of 30 NOD/LtSz-scid/scid mice vs 21 of 29 C.B-17/Sz-scid/scid animals. The multiple defects in innate and adaptive immunity unique to the NOD/LtSz-scid/scid mouse provide an excellent in vivo environment for reconstitution with human hematopoietic cells.

Mutations at the murine motheaten locus are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene.

Mice homozygous for the recessive allelic mutation motheaten (me) or viable motheaten (mev) on chromosome 6 develop severe defects in hematopoiesis. In this paper we present the findings that the me and mev mutations are within the hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. High resolution mapping localized me to an area tightly linked to Hcph on chromosome 6. Abnormalities of the Hcph protein product were demonstrated by Western blot analysis and by activity assays in both me/me and mev/mev mice. Molecular analysis of the Hcph cDNA identified abnormal transcripts in both mutants. DNA sequence analyses of cDNA and genomic clones revealed that both the me and mev mutations are point mutations that result in aberrant splicing of the Hcph transcript. These findings provide the first available animal models for a specific protein-tyrosine phosphatase deficiency, thus facilitating determination of the precise role of this signaling molecule in hematopoiesis.

Molecular genetic features reflecting the preference for isotype switching to IgA expression by Peyer's patch germinal center B cells.

It has proven difficult to evaluate the functional potential of germinal center (GC) B cells, including those from Peyer's patches (PP), by either in vivo or in vitro methods. Thus, rather than assess secreted Ig product as an indicator of functional potential we have instead sought to detect mRNAs related to the various Ig heavy chains in GC B cells from PP by in situ hybridization. We have found that the GCs of PP contain the vast majority of B cells with easily detectable levels of mRNA alpha. These levels are intermediate between those of small resting B cells and plasmablasts. When PP B cells are enriched for cells bearing GC markers, approximately 50% contain mRNA mu and 40% mRNA alpha. Similar enrichment for sIgA+ B cells gave 50% of cells with easily detectable mRNA alpha and few if any positive for mRNA mu. The sizes of these mRNAs were similar to those encoding the membrane and secretory form of mu and alpha chains. No C alpha germ-line transcripts could be detected by Northern analyses using a probe for sequences 5' to the alpha switch regions. Finally, GC and sIgA+ cells from PP also showed the absence of a portion of their genomic DNA for CH genes 5' of C alpha. Thus, it seems likely that most of the GC cells expressing mRNA alpha have undergone conventional VDJ recombination to C alpha at the DNA level in order to switch to the expression of IgA. Our findings reflect the extraordinary preference for switching to IgA by GC cells in PP.

Synthesis of abnormal immunoglobulins by hybridomas from autoimmune "viable motheaten" mutant mice.

Secretory defects in abnormal plasma cells, called Mott cells, that appear in lymphoid tissues of spontaneously autoimmune, "viable motheaten" (mev/mev) mice lead to deposition of immunoglobulin in RER-bound vesicles. Such vesicles have been termed Russel bodies. Cells with Russel bodies can also be observed rarely in normal animals, usually as a result of extreme antigenic loads or pathologic states. To understand why these abnormal cells appear commonly in mev/mev mice, we have established a panel of hybridomas that contain Russell bodies. Using immunochemical analysis and immunoelectron microscopy, we have characterized the secretory defects. Although these hybridoma cells synthesize a normal size heavy chain and it associates with light chain, the Russell bodies have many characteristics of inclusion bodies, which commonly appear in cells synthesizing mutant proteins and often are associated with incompletely or abnormally folded proteins. Pulse-chase experiments showed that immunoglobulins synthesized by these hybridomas accumulate rapidly into insoluble complexes and have an intracellular half life approximately 10 time greater than normal immunoglobulins. The defect affected only the immunoglobulin derived from the mev/mev mice and did not affect the secretion of normal immunoglobulin produced by an IgG1-secreting fusion partner. In addition to accumulating intracellular immunoglobulins, many mutant cell lines also secreted immunoglobulin. Endoglycosidase H digestion was used to determine the state of processing of the N-linked carbohydrates on the immunoglobulin molecules. This analysis demonstrated that the N-linked carbohydrates on the secreted immunoglobulin were resistant to endoglycosidase H digestion, indicating that they were processed normally. The insoluble IgM molecules were sensitive to endoglycosidase H, which is consistent with their localization to the RER. We propose several models by which these abnormal immunoglobulin-secreting cells commonly appear in this autoimmune mutant mouse.

Mutation in a reporter gene depends on proximity to and transcription of immunoglobulin variable transgenes.

Somatic mutation in immunoglobulin genes is localized to a 2-kilobase region of DNA surrounding and including rearranged variable (V), diversity, and joining (J) gene segments encoding heavy and light chains. To examine the structural basis for targeted mutation, we developed an assay to score mutation on plasmid substrates by using a reporter gene: a bacterial gene encoding an amber-suppressor tRNA molecule was placed 3' of a rearranged kappa VJ gene within the boundaries of mutation. The reporter gene is exquisitely suited for mutational analysis because it is only 200 base pairs (bp), which should not greatly disrupt structure of the immunoglobulin locus, and gene function depends on secondary structure, which means mutation can be scored in many different nucleotide positions. The plasmid was used to make transgenic mice, which were then immunized. The shuttle vector was retrieved by plasmid rescue into an indicator strain of Escherichia coli that contained an amber mutation in its beta-galactosidase gene. Integrity of the tRNA molecule was monitored by colony color, which permitted many transformants to be screened visually. Mutations were not seen in DNA from a transfected B-cell line grown in vitro or in DNA from nonlymphoid tissue of transgenic mice, indicating that the reporter gene was stable during cell division and DNA manipulations. However, when the transgenic mice were immunized, DNA from splenic B cells contained point mutations in the reporter gene at a frequency of 10(-3) per transformant. Sequence analysis of 17 mutated transgenes revealed that the mutations were 1- and 2-bp deletions in the tRNA gene, and one plasmid had an additional 2-bp deletion in the V gene. In contrast, previous studies have shown that mutations in endogenous VJ genes are predominantly nucleotide substitutions and have only 6% deletions. Two other plasmid constructs were analyzed in transgenic lines: no mutations were found when the tRNA gene was placed distal to the VJ gene, and no mutations were seen when the immunoglobulin promoter was deleted. Although we lack direct evidence that the deletions in the tRNA gene are caused by the same mechanism that acts on VJ genes, we have shown that mutations in this assay occur in a manner consistent with immunoglobulin-specific mutation in that they are found in splenic B cells and not in tail tissue, depend on position next to the VJ gene, and require transcription of the VJ gene.

Quality of antibodies secreted by clones in microcultures from B cells enriched on haptenated gelatin: isotypes and avidities.

Populations of murine B cells enriched for fluorescein (FLU)- or phosphocholine (PC)-binding cells stimulated with LPS, or FLU- or PC-LPS at low density in 10 microliter cultures form clones of cells that secrete antibodies. Antibody isotypes were determined by radioimmunoassay and their avidities were determined relative to standard, monoclonal antibodies by hapten inhibition using a radioimmunoassay. These analyses further characterize the development of B cell clones in microcultures and reveal that differing culturing conditions stimulate qualitatively different B cell populations to divide and differentiate. Without filler cells, isotype switching is rare. Co-culturing B cells with 10(5) (CBA/N x BALB/c) F1 male thymocyte filler cells leads to IgG and/or IgA antibody secretion by 15-20% of cultures; antibodies from clones that switch isotypes are exclusively of high avidity. IgM is almost always present as one clonal product; pre-switched cells rarely score in microcultures. Without filler cells, a high percentage of antibodies from FLU-LPS stimulated, FLU-binding cells are of high avidity (60%). However, clonotypes of lower avidity dominate with mitogenic culture conditions, 100 micrograms/ml LPS or with thymocytes. PC-binding cells are less sensitive to these mitogenic effects. Antibodies produced by PC-specific clones have a more restricted pattern of avidities and resemble in quality anti-PC antibodies produced in vivo.

CH isotype switching in B cell differentiation.

We have functionally defined a number of B cell subsets that likely represent B cells at different stages of development, based on the pattern of CH isotypes expressed by their clones in splenic fragment or microcultures and on those factors necessary in culture to support the growth of a clone displaying a particular isotype or set of isotypes. Our observations are consistent with isotype switching being a stochastic process which results in the occurrence of progressive isotype restriction in members of a diversifying clone. The surface marker best predictive of the pattern of isotypes a clone may secrete is the sIg isotype of its B cell precursor. Those B cells that have switched to the expression of non-IgM isotypes in vivo can be stimulated in vitro in splenic fragments to give an antibody-secreting clonal culture but so far cannot be stimulated in a microculture of dispersed cells that supports clones secreting IgM alone or with other isotypes.

Changes in specific B cells and the dissemination of the primed state in vivo following antigenic stimulation by different mucosal routes.

Immunologic dogma holds that the adaptive and long-term potential of the antibody response is fashioned by antigen-dependent, selective clonal proliferation of specific B cells and the retention of some which may undergo a second round of antigen-stimulated clonal expansion with antibody production. Apparently, the short-term, immediate consequences of an antibody response depend on the mix of isotypes displayed in vivo upon exposure to antigen. This latter seems to be clearly regulated by T cells, but it is also likely that the isotype potential of a B cell population and its future possible display of isotypes is linked to the initial, antigen-dependent proliferative phase in the development of an antibody response. In vitro analysis at limiting dilutions of specific B cells primed in vivo has led to the operational definition of IgA-committed cells. These B cells increase in frequency following chronic or acute antigenic stimulation of gut mucosa and have the potential to proliferate again in the presence of antigen and TH(Ag) cells to produce exclusively IgA. A general relationship exists between mucosal or parenteral priming of B cells and their potentials to express IgA and/or IgE--both isotypes appear to be likely products of secondary B cells and frequently both can be expressed by the same clone activated by a second-round of T-dependent antigenic stimulation. Cross priming--exposure of GALT or BALT leading to secondary B cells in the opposite mucosal lymphoid tissue--suggests an inherent antagonism between development of allergic (IgE) and putative allergy blocking (IgA) potentials.(ABSTRACT TRUNCATED AT 250 WORDS)