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Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited., Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 μM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

The rise of antimicrobial resistance (AMR) worldwide and the increasing spread of multi-drug-resistant organisms expressing metallo-β-lactamases (MBL) require the development of efficient and clinically available MBL inhibitors. At present, no such inhibitor is available, and research is urgently needed to advance this field. We report herein the development, synthesis, and biological evaluation of chemical compounds based on the selective zinc chelator tris-picolylamine (TPA) that can restore the bactericidal activity of Meropenem (MEM) against Pseudomonas aeruginosa and Klebsiella pneumoniae expressing carbapenemases Verona integron-encoded metallo-β-lactamase (VIM-2) and New Delhi metallo-β-lactamase 1 (NDM-1), respectively. These adjuvants were prepared via standard chemical methods and evaluated in biological assays for potentiation of MEM against bacteria and toxicity (IC50) against HepG2 human liver carcinoma cells. One of the best compounds, 15, lowered the minimum inhibitory concentration (MIC) of MEM by a factor of 32-256 at 50 μM within all tested MBL-expressing clinical isolates and showed no activity toward serine carbapenemase expressing isolates. Biochemical assays with purified VIM-2 and NDM-1 and 15 resulted in inhibition kinetics with kinact/ KI of 12.5 min-1 mM-1 and 0.500 min-1 mM-1, respectively. The resistance frequency of 15 at 50 μM was in the range of 10-7 to 10-9. 15 showed good tolerance in HepG2 cells with an IC50 well above 100 μM, and an in vivo study in mice showed no acute toxic effects even at a dose of 128 mg/kg.

BACKGROUND Fever of unknown origin and high sedimentation rate are common clinical problems. MATERIAL AND METHODS A middle-aged man with fever of unknown origin, night sweats and high sedimentation rate was referred to our hospital for investigation. RESULTS AND INTERPRETATION The patient was suspected to have mononucleosis or reactivation of infectious mononucleosis because of mild anaemia and thrombocytopenia, a weakly positive IgM antibody test for Epstein-Barr virus and monocytosis (in peripheral blood). Because monocytosis, elevated sedimentation rate and fever persisted, bone marrow smears were prepared and biopsies taken.The third biopsy showed that morphology was consistent with chronic myelomonocytic leukemia (CMML), which was confirmed by two later biopsies. However, a malignant cell population (consisting of blasts in peripheral blood) was only found in one of several flow cytometry assessments of peripheral blood and bone marrow aspirate and cytogenetic analyses of bone marrow cells were normal. The patient's clinical situation has been stable for some years and treatment has not been necessary.

A 70-year-old man with a seronegative rheumatoid arthritis showed some peculiar lamellar crystalline deposits mainly at the Descement/endothelial level in both eyes. Serum protein analysis revealed a monoclonal gammopathy with increased amounts of IgG and K-light chains along with traces of the latter in the urine. Corneal biopsy was not available, but electron microscopy of the conjunctival specimen demonstrated crystalline deposits within the endoplasmic reticulum of plasma cells lying within the connective tissue. This material is supposed to be of protein nature. It seems reasonable that the conjunctival deposits are identical to those seen in the cornea by slit-lamp examination, and it is also suggested that they represents paraprotein aggregates. After treatment with melphalan (Alkeran) and prednisone systemically, the corneal deposits decreased over a period of 9 months with corresponding improvement of the visual acuity.

In a randomized study of 92 previously untreated patients with multiple myeloma, the intention was to document the possible beneficial effect of combination chemotherapy including vincristine, carmustine, alkylating agents and prednisone, as compared to conventional therapy with melphalan and prednisone. Major prognostic factors did not differ significantly between the treatment groups. With the 2-drug therapy and 5-drug combination therapy, 48 and 54% of the patients achieved remission, respectively. Median survival for patients treated with the 2-drug regimen and 5-drug regimen was 29 and 33.5 months, respectively. No significant difference was found between the survival curves for stage III patients treated with the two regimens. After 12 months, patients who had achieved remission were randomized to have treatment discontinued or to have maintenance treatment. The numbers of relapses, remission duration and survival of the two groups were similar.

67 previously untreated patients with multiple myeloma were entered on a randomized clinical trial to determine whether the use of combination chemotherapy including vincristine, carmustine, alkylating agents, and prednisone was more effective than conventional therapy with melphalan and prednisone. The treatment groups did not show significant differences with respect to major prognostic factors. With the 2-drug combination therapy and 5-drug combination therapy, 67 and 74% of the patients achieved remission, respectively. Moreover, no significant difference was found between the two treatment schedules in terms of median survival (30 + months). The survival curves for stage III patients treated with the two regimens did not differ significantly. After 12 months, patients who had achieved remission were randomized to have treatment discontinued or to have maintenance treatment. 7 of 15 patients on maintenance therapy relapsed, whereas 9 of 14 patients who had their therapy discontinued relapsed, and the survival of the two groups was similar.

BACKGROUND Elevated haemoglobin and erythrocytosis are frequent causes for investigation. MATERIAL AND METHODS A 50-year-old woman with elevated haemoglobin and erythrocytosis was referred to our hospital for investigation. RESULTS AND INTERPRETATION She had elevated serum erythropoietin and iron deficiency. Mutational status of the JAK2 gene was negative with respect to polycythemia vera, and no secondary reasons for polycythemia - including tumours - were revealed. A bone marrow biopsy and bone marrow smear showed a moderately increased number of lambda- positive monoclonal plasma cells, and small amounts of lambda light chains were detected in the urine. Serum electrophoreses showed no gammopathy. The reason for her elevated haemoglobin could be an erythropoietin-producing tumour or an idiopathic erythrocytosis based on a mutation in exon 12 of the JAK2 gene. Investigation of her plasma cells revealed a 10-fold increase in erythropoietin mRNA expression, indicating the reason for her elevated serum erythropoietin and haemoglobin values.

To test the ability of pre-endoscopic clinical evaluation to predict clinically relevant findings of upper gastrointestinal endoscopy.Patients (341) who had been referred to upper gastrointestinal endoscopy for further evaluation of dyspeptic symptoms were included in this prospective, single-blinded study. Prior to endoscopy, the patients underwent a standardized clinical evaluation consisting of 1) a symptom questionnaire, 2) serological testing for Helicobacter pylori antibody and 3) determination of blood hemoglobin. Based upon this evaluation, patients were assigned to one of three defined risk groups. Group A comprised patients with known risk factors for diseases that would require further therapeutic or diagnostic management. Patients in groups B and C had no such risk factors. Patients in group C had heartburn or regurgitation as a predominant symptom, whereas patients in group B did not. The prevalence of clinically relevant findings upon upper endoscopy was then compared for these three groups.The prevalence of clinically relevant endoscopic findings in risk groups A, B and C were 20.1, 2.4 and 1.6%, respectively (p0.01 for both A versus B and A versus C). Furthermore, 89% of those with clinically relevant endoscopic findings belonged to group A, which comprised a total of 45% of the patients studied. In groups B and C, the prevalence of disease was similar to the area-specific prevalence in the general population without dyspeptic symptoms.By using a simple standardized questionnaire, H. pylori serology and a hemoglobin reading in the evaluation of dyspeptic patients under 45 years of age, the need for endoscopy can be reduced by 55%.

A 39-year-old man presenting with pulmonary infiltrations and hemolytic anemia was diagnosed as having primary sclerosing cholangitis (PSC) without evidence of ulcerative colitis. This constellation of associations is unique to the best of our knowledge. Autoimmune hemolytic anemia has been reported to be associated with PSC on only a few occasions, and pulmonary infiltrations in association with PSC are also quite unusual. Genetic and immunologic mechanisms are major factors in the pathogenesis of these disorders.

To evaluate how different methods for the detection of Helicobacter pylori influence on a "test, score and scope" decision approach in young dyspeptic patients.Complete data from 341 patients (52.2% males) were analyzed. One hundred and ten (32%) were H. pylori-positive by definition. The rapid serology test was true-positive in 64 patients, false-positive in 8, and false-negative in 46. For the EIA IgG serology test, the corresponding results were 99 true-positive, 7 false-positive, and 11 false-negative. If the H. pylori fecal test or urea breath test had been applied, 108 (98%) and 107 (97%) positives would have been correctly detected, respectively, as well as 14 and 7 false positives. Models using test data in a setting of decreasing H. pylori prevalence show that test properties have increasing significance.In a selection strategy for young dyspeptics based on the detection of H. pylori, the choice of test should be made with caution. H. pylori fecal test would probably give the best basis for such selection.

A woman in her fifties had been suffering for many years from iron deficiency anaemia as a result of unexplained heavy urinary iron loss (1). She had been receiving blood transfusions and intravenous iron supplements for 15 years, most recently in the form of large doses of iron (III) carboxymaltose. The woman then gradually developed pain extending from the lower back to both gluteal regions. Blood tests revealed normal calcium, low phosphate and elevated ALP levels, PTH in the upper normal range, normal 25-OH vitamin D and 1,25-(OH)2 vitamin D values. MRI scans (STIR sequence) detected high signal changes in both sacral wings and later also on the ileal side, indicating substantial bone oedema (left image). There were radiolucent lines in the sacral wings which raised suspicion of bilateral insufficiency fractures. Full body scintigraphy showed increased uptake in the anterior section of the frontal bone on both sides of the midline, in several ribs, both sacroiliac joints and proximally in the left tibia. Hypophosphataemic osteomalacia secondary to intravenous supplements of saccharated ferric oxide has been described previously (2). The small, electrically almost neutral iron saccharides are filtered in the glomeruli and impede phosphate reabsorption in proximal renal tubules. They also impede 1-alpha-hydroxylase activity and exacerbate the osteomalacia by directly inhibiting bone formation. Treatment consists of discontinuing iron saccharide supplements and optionally administering supplements of phosphate and vitamin D orally (2). Fortunately, the renal tubular dysfunction and osteomalacia appear to be reversible, but serious bone deformations may be permanent. At a check-up five months after termination of all iron supplements, the patient was recovering. MRI showed regression of pathological changes (right image). However, the patient still needs treatment for iron deficiency anaemia and is now receiving a different type of iron supplement intravenously. Her need for transfusion varies somewhat, depending on how often she receives iron.

The purpose was to review all patients with non-seminomatous germ cell tumours (NSGCT) treated at a single institution in order to evaluate the management and outcome. Patients were prospectively registered. Completed SWENOTECA forms and medical records of all 132 NSGCT patients treated between January 1985 and December 2000 were reviewed. Data on demographic, clinical, histological and biochemical characteristics as well as patient treatment and outcome were registered. The minimum follow-up was 2.2 years (median 8.3 years). In stage I, there was an overall relapse rate of 21%. These relapses were all treated successfully. Among stage II-IV patients, post treatment RPLND/surgical resections were performed in 31 patients (50%), and residual malignant disease was found in 23%. Relapse (N=3) in metastatic disease patients, were seen in stage IV only. In stage II-IV, 5 died from germ cell malignancy of whom 3 never achieved CR. Five-year overall and disease-specific survivals were 95% and 96%. For stage I, II, III, and IV, the 5-year disease-specific survivals were 100%, 98%, 100%, and 69% respectively. Grouped according to the International Germ Cell Consensus Classification, the 5-year overall survivals were 100%, 92%, and 60% for the good, intermediate, and poor prognosis groups of stage II-IV patients, respectively. This report is a complete review of NSGCT patients treated in our minor university clinic. Survival rates are comparable to recently published data, due to a commitment to multicentre protocol and research collaboration.

In this study we compared infrared tympanic thermometry with rectal mercury thermometry and digital rectal thermometry in patients admitted to a medical department. We found that infrared tympanic thermometry has a low sensitivity for detecting fever. Digital rectal thermometry is a good alternative to rectal mercury thermometry.

Prognostic factors have been tested in patients with multiple myeloma treated according to a randomized trial of standard therapy versus 5-drug combination therapy. The following population-based study included 92 patients with a median age of 70 yr. The median survival was 31 months. The Cox regression model was used to search for predictors of survival. The cut-off levels for blood analyses derived in earlier studies tended to select few patients in the high-risk groups, for example only 8% of the patients had hemoglobin (Hb) less than or equal to 7.5 g/dl. Lytic bone lesions in the pelvis or in the long bones, or spontaneous fractures and age greater than 70 yr gave prognostic information in addition to anemia and impaired renal function. The MRC staging system was a better prognostic tool than the Durie & Salmon stages. Palliative treatment regimens which take quality of life into account should be considered carefully in multiple myeloma patients greater than 70 yr.

The effect of activating human monocytes in vitro with recombinant Interferon-gamma (IFN-gamma) and lymphokines on monocyte-mediated cytotoxicity and on the production of cytotoxic protein factor(s) (CF), has been investigated. Lymphokines and IFN-gamma enhanced both cytotoxicity and CF production in a dose-dependent manner. A monoclonal antibody against human IFN-gamma abrogated the lymphokine-induced cytotoxic activity and CF production completely, indicating that the actual monocyte-activating factor in the lymphokine supernatant was IFN-gamma. At concentrations of 10(3)-10(4) U/ml, IFN-gamma induced an enhanced release of CF from the monocytes. However, IFN-gamma at concentrations (less than 100 U/ml) present in our lymphokine preparation was not sufficient to induce enhanced CF release. Since IFN-gamma at concentrations less than 100 U/ml activated monocytes for cytotoxicity, CF as an extracellular factor in the supernatant does not appear to be essential for monocyte-mediated cytotoxicity. However, using neutralizing antiserum raised against purified CF, indirect immunofluorescence microscopy revealed that IFN-gamma induced a marked accumulation of CF on the monocyte membrane. Taken together, the data shows that IFN-gamma is both necessary and sufficient at concentrations less than 100 U/ml for inducing monocyte-mediated cytotoxicity and production of CF as a membrane-associated component, but not as a released factor, suggesting that CF may function as a membrane-associated cytotoxin in monocyte-mediated cytotoxicity.

The contribution of monocyte cytotoxic protein factor (CF) to monocyte-mediated drug-dependent cellular cytotoxicity (DDCC) has been investigated. Cell lines which have been derived from murine WEHI 164 cells (termed WEHI 164 parental) by selecting for high (WEHI 164 clone 3) and low (R-WEHI 164) sensitivity to CF-mediated cytotoxicity were used as target cells in DDCC. By comparing the CF doses which produced 50% dead cells (LD 50) we found that WEHI 164 clone 3 was approximately 30 times more sensitive than WEHI 164 parental which in turn was 70 times more sensitive than R-WEHI 164. Actinomycin D (Act D) treatment of WEHI 164 parental and R-WEHI 164 greatly increase susceptibility to CF-mediated cytotoxicity. The susceptibility of WEHI 164 clone 3 was apparently somewhat increased at low dilutions of CF, whereas no significant increase was observed at high dilutions. The susceptibility to DDCC of the three target cell lines (WEHI 164 parental, WEHI 164 clone 3, and R-WEHI 164) correlated with the sensitivity pattern obtained in CF-mediated cytotoxicity of Act D-treated target cells. Monocyte- and CF-mediated cytotoxicity against Act D-treated WEHI 164 clone 3 and R-WEHI 164 was inhibited by neutralizing CF antiserum. These data indicate that CF is an effector molecule in monocyte-mediated DDCC.

WEHI 164 sarcoma cells cultured with monocyte-released cytotoxin (CF) for 4 weeks became resistant to CF-induced cytolysis and were concomitantly rendered resistant to monocyte-induced cytolysis. The resistant cell line (R-WEHI 164) has been stable with respect to resistance to monocyte- and CF-induced lysis for more than 7 months. WEHI 164 and R-WEHI 164 cells adsorbed CF and no significant difference in CF adsorption was observed. The results indicate that CF may be an effector molecule in monocyte-mediated cytolysis.

The effect of activating human monocytes in vitro with lipopolysaccharide (LPS) and muramyl dipeptide (MDP) on the production of cytostatic protein factor(s) (CF) has been investigated, and an antiserum against CF has been raised and tested. Upon incubation for 7 hr with LPS, in vitro differentiated human monocytes released CF. During LPS exposure, the presence of the protein synthesis inhibitor cycloheximide, at concentrations which reduced the overall protein synthesis by 60 and 80%, reduced the amount of CF released by only 20 and 40%, respectively. This indicates that the released CF was to a large extent already present in the monocytes before exposure to LPS. Compared to LPS, MDP induced only modest CF release. However, when lymphokine-activated monocytes were exposed to MDP, an increased CF release was observed. By immunizing a rabbit with CF purified by ion-exchange chromatography, chromatofocusing, and gel filtration, an antiserum was raised which neutralized the cytostatic activity released from monocytes exposed to LPS or lymphokines/LPS in sequence on the fourth day of culture. The cytostatic activity obtained by incubating freshly isolated monocytes with LPS was inhibited by the antiserum to a lesser extent, indicating the presence of other cytotoxins or cytotoxic cellular products in addition to CF in supernatants from freshly isolated monocytes. Various CF preparations were tested for IL-1 activity; no correlation between IL-1 activity and cytostatic activity was observed. Moreover, upon gel filtration the CF and IL-1 activities could be separated from each other and are consequently associated with different proteins.

The effect of activating human monocytes in vitro with lymphokines on the production of cytostatic protein factors) (CF) was investigated. Upon exposing the monocytes to either lymphokines or lipopolysaccharide (LPS) the amount of CF released was increased approximately fivefold compared to the amount released from unexposed monocytes. With sequential lymphokine and LPS treatment CF release increased nearly 10-fold. Even 10 min lymphokine activation before LPS exposure enhanced CF production significantly. The enhanced CF production was detected between 5 and 10 hr after lymphokine activation. The RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide reduced the lymphokine-induced CF production in a dose-dependent manner, indicating that lymphokines augment both CF mRNA and CF protein synthesis. When monocytes were exposed to LPS on both Day 2 and Day 4 of culture, the amount of CF obtained on Day 4 was reduced compared to that obtained on Day 2. A significant increase in CF production, however, was observed when the monocytes were activated with lymphokines before the second exposure to LPS on Day 4, supporting the view that lymphokines initiate synthesis of CF in monocytes. Upon ion exchange chromatography, chromatofocusing, and gel filtration the same elution profiles of CF were obtained irrespectively of whether the monocytes had been activated with lymphokines or not. This indicates that lymphokines induce an increased production of the same factor(s) which was obtained in the absence of lymphokines.

Human lymphokine-activated monocytes release a cytotoxic protein factor (CF). The cytostatic activity of CF was potentiated by the DNA and RNA synthesis inhibitors actinomycin D, daunomycin, and mitomycin C. These inhibitors increased the sensitivity for detecting CF approximately 10-fold, and this is of great practical value when assaying CF. The CF-potentialing inhibitors had a similar effect on the cell cycle distribution in that they all induced an accumulation of cells in the S and G2 phases of the cell cycle. Cytolytic activity was shown to be associated with CF, and this activity was also greatly potentiated by daunomycin and actinomycin D. The two other inhibitors studied, cycloheximide and 5-fIuorouracil (5-FU), had an adverse effect on the cytostatic activity of CF. These two inhibitors reduced the sensitivity of the assay for CF about fivefold. Cycloheximide had no apparent effect on the relative cell cycle distribution, whereas 5-FU induced an accumulation of cells in the G1 phase. Of the inhibitors studied, only those that induced an accumulation of cells in the S and G2 phases of the cell cycle potentiated the cytotoxic activity of CF. suggesting that CF may preferentially act in these parts of the cell cycle.