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Histone deacylase 11 and human sirtuins are able to remove fatty acid-derived acyl moieties from the ε-amino group of lysine residues. Specific substrates are needed for investigating the biological functions of these enzymes. Additionally, appropriate screening systems are required for identification of modulators of enzymatic activities of HDAC11 and sirtuins. We designed and synthesized a set of activity probes by incorporation of a thioamide quencher unit into the fatty acid-derived acyl chain and a fluorophore in the peptide sequence. Systematic variation of both fluorophore and quencher position resulted "super-substrates" with catalytic constants of up to 15,000,000 M -1 s -1 for human sirtuin 2 (Sirt2) enabling measurements using enzyme concentrations down to 100 pM in microtiter plate-based screening formats. It could be demonstrated that the stalled intermediate formed by the reaction of Sirt2-bound thiomyristoylated peptide and NAD + has IC 50 values below 200 pM., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Surgical indications for patients with pineal cysts are controversial. There are absolute indications such as hydrocephalus or tectal compression; otherwise, it is difficult to decide whether surgery would be beneficial when symptoms are not distinct., Objective: We tried to clarify the indications and clinical course of patients after resection of pineal cysts without ventriculomegaly., Methods: We reviewed our database for all patients operated on for pineal cyst without ventriculomegaly from 2003 to 2018. We studied the presenting symptoms, cyst size, surgical approach, extent of resection, and clinical and radiologic follow-up. Follow-up ranged from 3 months to 14 years (mean 3.74 years)., Results: Forty-three patients underwent surgery for pineal cyst in absence of ventriculomegaly; 36 female patients, 7 male patients, mean age 25.63 years (±10.62 years, range 4-52 years). Presenting symptoms included headache (41/43), nausea and vomiting (31/43), dizziness (19/43), visual disturbances (10/43), and sleep disturbances (8/43). Mean cyst size was 15.7 mm (±4.9 mm, range 9-27 mm). In total, 41 of 43 patients reported a good outcome, represented by a Chicago Chiari Outcome Scale score of 11 or greater and only 2 of 43 patients reported a bad outcome, defined by Chicago Chiari Outcome Scale score of 10 or less., Conclusions: We suggest that pineal cysts without ventriculomegaly are an indication for surgery when patients present with headache and/or visual disturbances and other causes have been excluded. Resolution of symptoms and quality of life in our cohort may denote a good indication for resection. However, we must admit that there is still no evidence to recommend this technique as a treatment of headache in these patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: The main objective of the present prospective, randomized, single-blinded controlled study was to measure heat during bony decompression of lumbar spinal stenosis with high-speed drills and an ultrasonic bone-cutting knife., Methods: Ninety patients diagnosed with lumbar spinal stenosis were included in this study and randomized for lumbar spinal canal decompression using either a high-speed drill with automatic irrigation, high-speed drill with manual irrigation, or an ultrasonic bone-cutting knife with automatic irrigation (USBCD). For evaluation of group homogeneity, a visual analog scale pain score and neurologic findings were measured preoperatively and postoperatively. Temperatures during bony decompression were measured using a forward-looking infrared camera system., Results: Clinical results among the 3 groups did not differ in pain reduction, improvement of neurologic findings, or the rate of complications. However, significantly lower values were found for absolute and mean maximal temperatures during bony decompression in the USBCD group compared with the groups of patients who received the high-speed drill with automatic irrigation and the high-speed drill with manual irrigation, indicating this technique to be less aggressive in terms of thermal induction of bone necrosis. USBCD allows more precise bone removal compared with high-speed drills, and despite increased device time, no significant difference in the overall decompression time was observed., Conclusions: All methods examined produced short temperature peaks with possible, at least temporary, damage to bone and neural tissue. Automatic irrigation was associated with lower heat development compared with manual irrigation. Despite evidence of critical temperatures, no clinical correlation (e.g., neurologic deficits) was encountered in this study. Clinically, all 3 methods presented equally good results., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Oral and Maxillofacial operations can be extremely frightening treatment situations. General data on this phenomenon should be acquired through a multi-centre trial., Method: A 10-page questionnaire was developed which was answered by 600 oral and maxillofacial patients before individual treatment. Control groups consisted of 800 surgical, dental and general practitioners/internal medicine patients. Twenty six medical practices and hospital departments in 11 German cities were involved in the trial., Results: Both subjective anxiety assessment by patients and objective testing (State Trait Anxiety Inventory; STAI) indicated a high level of treatment anxiety for oral and maxillofacial treatment situations. Oral and Maxillofacial Surgery (OMFS) patients' scores were significantly higher than those of control groups. Especially younger and female patients, patients treated under local anaesthesia and out-patients showed intensive anxiety. Previous treatment experience in OMFS did not modify test results., Conclusion: Managing perioperative anxiety is still a major challenge in OMFS. Irrespective of technical, pharmacological and surgical advances, developing and establishing stress-reducing and anxiolytic perioperative techniques is of considerable importance for both patients and surgeons.

Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract illness in infants. However, the mechanisms leading to resolution of RSV infections are poorly understood. Since alveolar macrophages play an important role in defending the respiratory tract against infectious agents we investigated the interactions of RSV with these cells. Murine alveolar macrophages were challenged in vitro with RSV at different multiplicities of infection. The percentage of macrophages expressing viral antigen was determined by staining with monoclonal anti-RSV antibodies and evaluation by fluorescence microscopy or FACS analysis. The ability of macrophages to support virus replication was measured by a plaque forming assay on HEp-2 cells. Cell lysates of macrophages contained only small amounts of viable RSV in comparison to disrupted HEp-2 cells. The amount of viable RSV as well as the percentage of macrophages expressing viral antigen decreased rapidly over time. Activated macrophages had a reduced virus load in comparison to resting macrophages. RSV infected macrophages released biologically active tumor necrosis factor (TNF) in a virus dose dependent manner. In contrast, a high virus inoculum resulted in reduced microbicidal activity and oxygen radical production. Our results suggest that RSV infection influences different functions of alveolar macrophages in various ways. Since TNF is thought to restrict viral replication in several cell types it may play a role in limiting virus replication.

Analysis of the functional activity of phagocytes is of great importance in the differential diagnosis of patients with recurrent bacterial infections. Here we describe a method to determine the production of reactive oxygen intermediates (ROI) by microcytofluorometry using dihydrorhodamine 123, a derivative of rhodamine 123. Using this method the ROI production of erythrocyte-depleted whole blood samples can be measured without further time-consuming purification steps. Possible harmful manipulation of the isolated cells can also be avoided and highly reproducible and significant results are obtained in the minimum of time. This assay provides a very sensitive alternative to the clinically used NBT test in the diagnosis of patients with chronic granulomatous disease (CGD). Moreover, the analysis of oxygen-dependent effector functions of murine effector cells and cell lines may be important in investigating resistance to certain microbes (e.g., Candida albicans, Staphylococcus aureus or different protozoa such as Toxoplasma gondii or Leishmania species).

Supernatants from Concanavalin A-stimulated murine spleen cells were subjected to hydrophobic interaction chromatography on phenyl-Sepharose. Macrophage cytotoxicity factor (MCF), macrophage migration inhibitory factor (MIF), T-helper cell-replacing factor (TRF) and colony-stimulating factor (CSF) were bound at high ionic strength and were released stepwise at low ionic strength. CSF thus could be separated from MCF, MIF and TRF and the bulk of other proteins. Chromatograhy of pools containing MCF, MIF and TRF on Sephadex did not lead to a separation of the three activities which were all found in a molecular weight range of 25.000-55.000. Isoelectric focusing of these pools in pH range from 4 to 9 gave two peaks for MCF in a single sharp peak at pH 5.3. The results demonstrate that the four biological activities can be distinguished on a chemical basis and are accessible for purification and chemical characterization.

Cells of the mononuclear phagocyte system can be activated by lymphokines to both increased extracellular cytotoxicity against tumor targets and intracellular cytotoxicity against micro-organisms. In addition, these effector cells can kill antibody-coated target cells in an ADCC system. These two cytotoxic mechanisms can co-operate and act synergistically. Such an synergistic action is characterized by the specificity of the antibody which coats the target cells and not by the non-specific activation induced by high dosages of lymphokine. The lymphokine MCF has partially been purified and separated from a variety of other lymphokines. This purified material, when injected into mice intraperitoneally, activates the macrophages to strong cytotoxicity. Evidence obtained by the use of different rat anti-mouse macrophage monoclonal antibodies suggests that there exist different subpopulations of macrophage and that some of these subpopulations can be correlated to defined functions.

We previously reported that highly purified bone marrow-derived macrophage precursors can exert strong spontaneous cytotoxicity against YAC-1 tumor cells, Candida albicans, and protozoa of the genus Leishmania. In the present paper, evidence is shown that macrophage precursors in normal untreated mice are not confined to the bone marrow compartment but can also be found in the spleen. These organ-associated cells, which have the same buoyant density as large granular lymphocytes, have been positively sorted by means of an indirect rosetting technique employing the macrophage-specific monoclonal antibodies F4/80 and M143. The rosetting fractions represented an extremely homogeneous population of macrophage precursors characterized by high candidacidal and natural killer activity and by a strong proliferative response to the macrophage-specific colony-stimulating factor CSF-1. Spleen- and bone marrow-derived macrophage precursors differed in their target selectivity. In addition, the mature macrophages derived in vitro from these two precursor populations displayed striking differences in their candidacidal activity. The implications of these findings in relationship to heterogeneity in the macrophage differentiation line are discussed.

A simple way of measuring and evaluating lactate dehydrogenase release from lysed tumor cells is described. LDH activity was determined as NADH oxidation or INT reduction over a defined time interval, which was limited by stopping the enzymatic reaction with the inhibitor oxamate. Reaction products were then assayed using a microplate reader. The principle of measuring LDH activity of cellular culture supernatants as a measure of cytotoxicity was successfully applied to a number of murine and human effector-target cell combinations (macrophages, monocytes, NK cells and cytotoxic T cells with P815, A375, K562 and Yac-1 tumor cells) as well as to the determination of TNF activity on L929 cells. Comparison with 51Cr release assays suggests that LDH release assays are an appropriate and possibly preferable means of measuring cellular cytotoxic reactions. This LDH release assay combines the advantages of reliability and simple evaluation characteristic of radioisotope release assays with the convenience of speed and avoidance of radioactivity.

Since March, 1983, in vitro tests for the quantitative determination of the tumor-associated antigen Ca 19-9 (carbohydrate antigen 19-9) were performed in more than 300 patients with confirmed tumors. These tests had as a goal determination of the diagnostic value of this new potential tumor marker Ca 19-9 with regard to specificity and sensitivity of malignant growths in the gastrointestinal tract, breast, and lung. The applicability of Ca 19-9 RIA in posttherapeutic case control for early registration of recurrences and metastases was examined. First analyses of our patient collective confirmed the assumption that increased serum concentrations of Ca 19-9 are detectable in gastrointestinal tumors as well as in mammary and bronchial carcinoma. Thus Ca 19-9 is a nonspecific tumor-associated antigen that by itself does not allow an organ correlation of malignancies of the gastrointestinal tract, breast, and lung.

The in vitro differentiation and maturation of resident and activated mouse and human macrophages (M phi) from different anatomical sources was investigated with regard to their oxygen metabolism during zymosan phagocytosis. We found evidence that chemiluminescence (CL) of M phi depends upon their differentiation stage: a) In the absence of any phagocytic stimulus, the human M phi showed a lucigenin-dependent CL background that was approximately 10-fold higher than in mouse M phi and decreased to low levels in resident M phi (monocyte-derived human M phi). This background was reduced by SOD to about 50%. No relevant luminol-dependent background was observed in all mouse and human M phi during culture time. b) Resident and activated mouse and human M phi could be distinguished in terms of their lucigenin-dependent CL during zymosan phagocytosis, which was persistently high in activated M phi, but decreased to comparatively low levels in resident M phi during culture time. This zymosan-elicited CL was almost completely SOD-dependent during all culture time. c) A dissociation between phagocytosis and oxygen radical release is observed: the decrease of both minolul and lucigenin-dependent CL in resident phagocytizing M phi during maturation did not correspond to a decrease of their phagocytic activity. Phagocytosis occurred at a high rate also in the absence of a relevant CL-detectable generation of oxygen radicals. The oxygen radical release, as measured by SOD-inhibitable cytochrome c reduction, paralleled CL during zymosan phagocytosis and declined with maturation of monocytes into M phi. In contrast, the zymosan-induced nitro-blue-tetrazolium reduction increased in mature resident human M phi. Thus, it seems that different metabolic pathways are utilized during phagocytosis in young and mature M phi.

A solid-phase immunoradiometric assay is described for the detection of mouse immunoglobulin classes and subclasses in unpurified and unconcentrated supernatants of hybridomas. IgG fractions from rabbit antisera specific for mouse immunoglobulin classes and subclasses are used for coating the wells of flexible microtiter plates. Monoclonal antibody present in hybridoma supernatants is bound only to wells that contain the appropriate anti-subclass antibody. The binding of hybridoma antibodies to corresponding IgG subclasses or IgM is then detected by a labeled rabbit anti-mouse antibody binding to all mouse immunoglobulins (heavy and light chains). Thus, only 1 labeled antibody is needed for all assays. The advantages of the method described are the following: results are obtained within a few hours and antibody containing hybridoma supernatants may be used without a concentration step since minute amounts of antibody are detected by the immunoradiometric assay. Cultures producing several subclasses may be early recognized as oligo/polyclonal.

Cooperative effects are described between 2 different mechanisms of macrophage-mediated cytotoxicity, the lymphokine-induced and the antibody-dependent cytotoxicity:Macrophages are activated to nonspecific cytotoxicity by a 24 h incubation with lymphokines. Young macrophages cultured from the bone-marrow or obtained from induced peritoneal exudates, but not the resident peritoneal macrophages, have the capacity to kill antibody-coated tumor cells. To obtain a cooperative effect between the 2 mechanisms both the lymphokine and the antibody have been titrated to a dilution, at which the macrophages did not exert any cytotoxicity. At such subthreshold dosages, however, a very good synergistic effect between the 2 mechanisms was observed. This synergism was obtained with bone-marrow macrophages and with thioglycollate-induced peritoneal macrophages. Macrophages of C3H/HeJ mice, which have been reported to be a low responder strain for lymphokine activation, showed synergistic effects with similar lymphokine concentrations as macrophages from the high responder strain C57BL/10. Thus, in this synergistic situation the low responsiveness of C3H/HeJ mice is completely compensated. Since during an immune reaction in most cases lymphokine as well as antibody are produced, this synergistic reaction will be of particular relevance for in vivo-situations.

C57BL/6 macrophage populations from spleen and liver, the main organs for the manifestation of visceral leishmaniasis, were investigated for their ability to perform spontaneous phagocytosis-associated killing of 3H-thymidine (3H-TdR)-prelabelled L. donovani amastigotes and promastigotes. The results showed that organ macrophages from spleen and liver killed L. donovani amastigotes and promastigotes spontaneously with high efficiency. This consistent finding was first detectable at 2-3 h, and the reaction was completed at 12 h. This type of killing was strongly enhanced when spleen and liver macrophages were activated. This phagocytosis-associated killing mechanism may contribute, to a large extent, in maintaining the infection under control in vivo, by drastically reducing the amount of parasites that is required to establish intracellular parasitism. To be able to assay phagocytosis-associated destruction of both promastigotes and amastigotes, a reproducible system for the production in vitro of Leishmania donovani amastigotes by the macrophage cell-line J774 was developed. The DNA of the Leishmania amastigotes was labelled with 3H-TdR with high efficiency. The spontaneous label release of prelabelled L. donovani amastigotes was comparable to that of prelabelled promastigotes over an assay period of 24 h.

An in vitro radiometric assay is described for the detection of cytolytic activity against the obligate intracellular parasite Leishmania enriettii. The assay system can be equally well applied to non-cellular (humoral), cellular non-phagocytic, and cellular phagocytic effector situations. Leishmania promastigote organisms are DNA-labeled with [methyl-3H]thymidine [( 3H]dThd) with high efficiency. Spontaneous label release remains very low even in non-ideal culture conditions. We have modeled a leishmanicidal situation by co-cultivating L. enriettii with starch-elicited murine peritoneal macrophages for different time periods. At an effector-to-target ratio of 1:6 a highly significant [3H]dThd release can be observed after 4 h of co-cultivation, reaching 60% after 18 h. The principle advantages of this assay system are speed, high sensitivity and objectivity. This makes it suitable for mass screening of, e.g., immunomodulatory or parasiticidal agents and equally useful in both phagocytic and non-phagocytic situations.