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Background: Management of insulin administration for intake of carbohydrates and physical activity can be burdensome for people with type 1 diabetes on hybrid closed-loop systems. Bihormonal fully closed-loop (FCL) systems could help reduce this burden. In this trial, we assessed the long-term performance and safety of a bihormonal FCL system., Methods: The FCL system (Inreda AP; Inreda Diabetic, Goor, Netherlands) that uses two hormones (insulin and glucagon) was assessed in a 1 year, multicentre, prospective, single-arm intervention trial in adults with type 1 diabetes. Participants were recruited in eight outpatient clinics in the Netherlands. We included adults with type 1 diabetes aged 18-75 years who had been using flash glucose monitoring or continuous glucose monitors for at least 3 months. Study visits were integrated into standard care, usually every three months, to evaluate glycaemic control, adverse events, and person-reported outcomes. The primary endpoint was time in range (TIR; glucose concentration 3·9-10·0 mmol/L) after 1 year. The study is registered in the Dutch Trial Register, NL9578., Findings: Between June 1, 2021, and March 2, 2022, we screened 90 individuals and enrolled 82 participants; 78 were included in the analyses. 79 started the intervention and 71 were included in the 12 month analysis. Mean age was 47.7 (SD 12·4) years and 38 (49%) were female participants. The mean preintervention TIR of participants was 55·5% (SD 17·2). After 1 year of FCL treatment, mean TIR was 80·3% (SD 5·4) and median time below range was 1·36% (IQR 0·80-2·11). Questionnaire scores improved on Problem Areas in Diabetes (PAID) from 30·0 (IQR 18·8-41·3) preintervention to 10·0 (IQR 3·8-21·3; p<0·0001) at 12 months and on World Health Organization-Five Well-Being Index (WHO-5) from 60·0 (IQR 44·0-72·0) preintervention to 76·0 (IQR 60·0-80·0; p<0·0001) at 12 months. Five serious adverse events were reported (one cerebellar stroke, two severe hypoglycaemic, and two hyperglycaemic events)., Interpretation: Real-world data obtained in this trial demonstrate that use of the bihormonal FCL system was associated with good glycaemic control in patients who completed 1 year of treatment, and could help relieve these individuals with type 1 diabetes from making treatment decisions and the burden of carbohydrate counting., Funding: Inreda Diabetic., Competing Interests: Declaration of interests HB, TJPJ, MK, and MW are employees of Inreda Diabetic. GDL has received consulting fees from AstraZeneca, Boehringer Ingelheim, and Lilly, and payment or honoraria from Sanofi, and AstraZeneca; was a member of the scientific Advisory Board of Dutch Diabetes Fund; received support for attending meetings or travel from Sanofi; and has received institutional grants from Boehringer Ingelheim, Sanofi, and ZGT Research Fund. MO was a board member of De Diabeteskamer, the association of Dutch diabetes internists. TU has provided consultancy services to Dexcom and has received honorariums for lectures from Medtronic, Dexcom, Sanofi, and Inreda Diabetic. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

In Northwestern Patagonia (Chile), three species of Morchella from undisturbed environments have been identified to date: Morchella tridentina, Morchella andinensis and Morchella aysenina, all belonging to the Elata clade and associated mainly with Nothofagus forests. In this study, the search for Morchella specimens was extended to disturbed environments in Central-Southern Chile, to further explore Morchella species diversity in the country, which is still very limited. The Morchella specimens were identified through multilocus sequences analysis, and the mycelial cultures were characterized, establishing comparisons with specimens from undisturbed environments. To the best of our knowledge, these results reveal for the first time in Chile the presence of the species Morchella eximia and Morchella importuna, and in the case of the last one also the first record in South America. These species were found associated almost exclusively with harvested or burned coniferous plantations. The in vitro mycelial characterization revealed certain inter- and intra-specific patterns of the morphology, such as pigmentation, mycelium type, and development and formation of sclerotia, which varied according to growth media and incubation temperature. The growth rates (mm/day) and mycelial biomass (mg) were significantly influenced by the temperature (p < 0.05), with maximum rates (>10 mm/day) and biomass (approx. 20 mg) between 20 and 24 °C, while a significant growth reduction (70-90%) was observed at 28 °C, mainly in the species from undisturbed environments. Potato-dextrose (PDA) medium stimulated the greatest mycelial density and sclerotia formation in most of the isolates, mainly in M. eximia (UDEC-LAF 236 isolate) which recorded the best mycelial growth performance. Among isolates, UDEC-LAF 236 also showed the best performance in sclerotia production (>350 sclerotia/dish) in 10 days of growth. This study contributes to the knowledge of the diversity of Morchella species in Chile by broadening the species range to those from disturbed environments. It also provides molecular and morphological characterization of the in vitro cultures of different Morchella species. The report on M. eximia and M. importuna, species known as cultivable, adapted to local climatic and edaphic conditions could represent the first step to developing artificial Morchella cultivation methods in Chile., Competing Interests: Declaration of competing interest The author declares that they have no conflict of interest for this publication., (Copyright © 2023 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Silverleaf is caused by the fungus Chondrostereum purpureum , which produces wood necrosis and foliar silvering in woody plants. Field observations and studies in apple have shown the reversion of foliar symptoms. Because plants were clones and received identical agronomical management, it was hypothesized that reversion is driven by endophytic microbiota. Thus, the objectives of this study were to compare healthy, diseased, and reverted plants with respect to their physiology, endophytic microbial communities, antagonistic ability of their endophytes against C. purpureum, and defense genes expression. Water potential, stomatal conductance, chlorophyll content, and fluorescence were measured. Endophytic bacterial and fungal DNA were analyzed by denaturing gradient gel electrophoresis, and community richness and similarity were calculated. Wood cores were collected and bacterial and fungal endophytes were isolated and confronted with C. purpureum- virulent strains in dual-culture assays. Defense genes expression was measured by quantitative PCR. Results indicated that there were no differences in physiological parameters between healthy and reverted plants, except for fluorescence, and both type of plants differed from diseased ones. Bacterial and fungal community richness was similar in healthy and reverted plants and higher than in diseased ones. Endophytes from reverted and healthy plants showed high antagonism to C. purpureum . Furthermore, nonexpressor of pathogenesis-related gene 1 expression was upregulated in reverted plants, whereas phenylalanine ammonia lyase and polygalacturonase-inhibiting protein genes showed higher values in diseased plants. Overall, physiological, molecular, and microbial characteristics were similar between healthy and reverted plants, and both differed from diseased ones. Therefore, reversion of symptoms is associated with changes in the endophytic microbiota, which seems to be a promising source of biological control agents against C. purpureum .

The phenotypic diversity and productivity of a diverse alfalfa ( M. sativa subspp.) panel of cultivars, landraces and wild relatives with putative drought tolerance were evaluated in two Mediterranean environments (central Chile and Southern Australia). In Chile, 70 accessions were evaluated in rainfed conditions and in Australia 30 accessions under rainfed and irrigated conditions, during three growing seasons. Large phenotypic variation was observed among and within subspecies for NDVI, stem length, intercepted PAR and forage yield. Principal component analysis indicated that the first two principal components (PC) accounted for 84.2% of total variance; fall dormancy, taxa, and breeding status were closely related to the agronomical performance of alfalfa accessions. Forage yield varied largely among accessions across years and locations. A linear relationship was found between annual forage yield and annual water added to the experiments (R 2 = 0.60, p < 0.001). The GxE analysis for forage yield allowed the detection of the highest yielding accessions for each of the two mega-environments identified. The accessions CTA002 and CTA003 showed greater forage yield in both Chile and Australia environments. It is concluded that new breeding lines derived from crosses between cultivated alfalfa ( M. sativa subsp. sativa ) and wild relatives belonging to the primary ( M. sativa subsp. falcata ) and tertiary ( M. arborea ) gene pool, achieve outstanding agronomical performance in drought-prone environments.

Objective: Type 1 diabetes (T1D) is characterised by islet autoimmunity and beta cell destruction. A gut microbiota-immunological interplay is involved in the pathophysiology of T1D. We studied microbiota-mediated effects on disease progression in patients with type 1 diabetes using faecal microbiota transplantation (FMT)., Design: Patients with recent-onset (<6 weeks) T1D (18-30 years of age) were randomised into two groups to receive three autologous or allogenic (healthy donor) FMTs over a period of 4 months. Our primary endpoint was preservation of stimulated C peptide release assessed by mixed-meal tests during 12 months. Secondary outcome parameters were changes in glycaemic control, fasting plasma metabolites, T cell autoimmunity, small intestinal gene expression profile and intestinal microbiota composition., Results: Stimulated C peptide levels were significantly preserved in the autologous FMT group (n=10 subjects) compared with healthy donor FMT group (n=10 subjects) at 12 months. Small intestinal Prevotella was inversely related to residual beta cell function (r=-0.55, p=0.02), whereas plasma metabolites 1-arachidonoyl-GPC and 1-myristoyl-2-arachidonoyl-GPC levels linearly correlated with residual beta cell preservation (rho=0.56, p=0.01 and rho=0.46, p=0.042, respectively). Finally, baseline CD4 +CXCR3+T cell counts, levels of small intestinal Desulfovibrio piger and CCL22 and CCL5 gene expression in duodenal biopsies predicted preserved beta cell function following FMT irrespective of donor characteristics., Conclusion: FMT halts decline in endogenous insulin production in recently diagnosed patients with T1D in 12 months after disease onset. Several microbiota-derived plasma metabolites and bacterial strains were linked to preserved residual beta cell function. This study provides insight into the role of the intestinal gut microbiome in T1D., Trial Registration Number: NTR3697., Competing Interests: Competing interests: MN and WMDV are founders and in the Scientific Advisory Board of Caelus Health, the Netherlands. WMDV is Founder and in the Scientific Advisory Board of A-Mansia, Belgium. MN is in the Scientific Advisory Board of Kaleido Biosciences, USA., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Silverleaf caused by the basidiomycete Chondrostereum purpureum affects numerous woody species, including fruit tree crops like apple, resulting in wood necrosis and foliar silvering. There are no curative alternatives for this disease, and its management is by prevention methods. Therefore, the aim of this study was to develop a rapid diagnostic tool for the detection and identification of C. purpureum directly from woody tissues to help distinguish the pathogen from other basidiomycetes that are commonly found on apple. The silverleaf pathogen was isolated from different hosts and locations, and Koch's postulates were performed by inoculating the isolates on apple cuttings and measuring internal necrosis. A previously described APN 1 pair of primers specificity was also tested against 25 C. purpureum isolates in this study, using other wood rotting species as negative controls. Seven virulent isolates were inoculated on apple cuttings, and DNA was extracted from the cuttings' sawdust and amplified using APN 1, after 22 days of incubation. To prove the efficiency of the method in the field, DNA from healthy nursery plants inoculated with two virulent isolates, and naturally infected plants showing different levels of foliar symptoms, were tested. Presence of the fungus was verified by reisolation on APDA in all assays. Koch's postulates indicated that all C. purpureum isolates were pathogenic, showing different virulence levels, and APN 1 primers were able to discriminate them from other basidiomycetes. The method was also able to detect C. purpureum from artificially inoculated plants as well as naturally infected ones, demonstrating that the protocol may become a rapid minimally destructive diagnostic tool to detect the pathogen without the need to isolate it from tissues, and thus taking measures to prevent its dissemination.

Aim: The molecular typing and the susceptibility of Staphylococcus aureus strains of swine origin to antibiotics, oregano (Origanum vulgare L.) essential oil (EO) and Chilean blackberry maqui (Aristotelia chilensis (Molina) Stuntz) extract were determined., Methods and Results: Twenty S. aureus strains of swine origin were subjected to molecular typing, of which six strains were selected for antimicrobial susceptibility testing. The epsilon test (Etest) was used to determine the antibiotic susceptibility. The susceptibility to natural antimicrobials (NAs): oregano EO, maqui extract, thymol (Thy) and carvacrol (Carv), was carried out using the disk diffusion method. The S. aureus strains were genetically diverse. All strains were resistant to at least one class of antibiotic, and two strains were multidrug-resistant. The minimum inhibitory concentration of oregano EO, Thy and Carv was 0·01-0·04%. Maqui extract did not show antistaphylococcal activity., Conclusions: Natural antimicrobials extracted from oregano have an inhibitory activity against S. aureus strains from swine origin, with no effect using maqui extract., Significance and Impact of the Study: This study provides information about the characteristics of S. aureus strains of swine origin, and about the potential use of NAs from oregano to enhance the control of antibiotic-resistant S. aureus strains in the pork supply chain., (© 2019 The Society for Applied Microbiology.)

The conserved DnaA-oriC system is used to initiate replication of primary chromosomes throughout the bacterial kingdom; however, bacteria with multipartite genomes evolved distinct systems to initiate replication of secondary chromosomes. In the cholera pathogen, Vibrio cholerae, and in related species, secondary chromosome replication requires the RctB initiator protein. Here, we show that RctB consists of four domains. The structure of its central two domains resembles that of several plasmid replication initiators. RctB contains at least three DNA binding winged-helix-turn-helix motifs, and mutations within any of these severely compromise biological activity. In the structure, RctB adopts a head-to-head dimeric configuration that likely reflects the arrangement in solution. Therefore, major structural reorganization likely accompanies complex formation on the head-to-tail array of binding sites in oriCII. Our findings support the hypothesis that the second Vibrionaceae chromosome arose from an ancestral plasmid, and that RctB may have evolved additional regulatory features., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The dissociative recombination of CaO + ions with electrons has been studied in a flowing afterglow reactor. CaO + was generated by the pulsed laser ablation of a Ca target, followed by entrainment in an Ar + ion/electron plasma. A kinetic model describing the gas-phase chemistry and diffusion to the reactor walls was fitted to the experimental data, yielding a rate coefficient of (3.0 ± 1.0) × 10 -7  cm 3  molecule -1  s -1 at 295 K. This result has two atmospheric implications. First, the surprising observation that the Ca + /Fe + ratio is ~8 times larger than Ca/Fe between 90 and 100 km in the atmosphere can now be explained quantitatively by the known ion-molecule chemistry of these two metals. Second, the rate of neutralization of Ca + ions in a descending sporadic E layer is fast enough to explain the often explosive growth of sporadic neutral Ca layers.

Severe hepatitis due to herpes simplex virus type 1 (HSV-1) in immunocompetent patients is a very rare event. The acute hepatitis may lead to fulminant deterioration of liver function and can be rapidly fatal. The diagnosis should be considered in case of severe hepatitis of unknown cause. Early consideration of HSV-1 hepatitis in the differential diagnosis in an adult patient, also with an apparently normal immune system, is important and early initiation of antiviral treatment may be lifesaving in this situation.

Background: Type B lactic acidosis is thought to be a rare complication of malignancy. It was first described in patients with acute leukaemia by Field et al. in 1963. Since then, it has been observed more often, in particular in haematological malignancies and rarely in solid tumours., Methods: Previously reported cases of lactic acidosis in solid malignancy are reviewed. In addition, we report a case of type B lactic acidosis in a woman with metastatic breast cancer. Afterwards, we speculate on the elusive pathophysiology of this oncological emergency., Results: 14 cases of lactic acidosis due to solid malignancies, without prior chemotherapy, were identified. The cases were published from the year 1978 to 2006., Discussion: Several theories concerning the mechanism for type B lactic acidosis in solid malignancy have been postulated. During the last decade, more and more evidence supports the role of overproduction of lactic acid due to ischaemia in the neoplastic tissue bed and with cancer cells having an aberrant energy production.

Objective: The concept of disease activity in Graves' ophthalmopathy (GO) might explain why as many as one-third of patients do not respond to immunosuppressive treatment, because only patients in the active stage of disease are expected to respond. The hypothesis was adopted that a parameter used to measure disease activity should be able to predict a response to immunosuppressive treatment. The aim of this study was to develop a multivariate prediction model in which all previous tested activity parameters are integrated., Design and Patients: We included 66 consecutive patients with untreated moderately severe GO who had been euthyroid for at least 2 months. All patients were treated with radiotherapy. Measurements Treatment efficacy after 6 months follow-up was used as the primary outcome measure. Disease severity and 15 different disease activity parameters were assessed before treatment. Univariate and multivariate logistic regression models were used to predict response (model 1) or no change (model 2)., Results: In multivariate analyses, we found that duration of GO, soft tissue involvement, elevation, soluble interleukin-2 receptor (sIL-2R), soluble CD30 (sCD30), eye muscle reflectivity and octreotide uptake ratio were significant predictors of a response to radiotherapy. Gender, duration of GO, soft tissue involvement, eye muscle reflectivity, IL-6 and urinary glycosaminoglycan (GAG) excretion were significant predictors of no change upon radiotherapy. Prognostic score charts were developed for use in clinical practice to calculate the probability of response (model 1) and the probability of no change (model 2) for each new patient. Finally we used a combination of both models to define a recommended treatment modality for each individual patient, based on both the predicted probabilities of response and no change. We were able to identify the correct treatment (based on a comparison with the observed response) in 89% of the patients., Conclusions: Although we strongly recommend that our results should be confirmed in other studies, our findings are the first evidence for the idea that disease (in)activity should determine which kind of treatment should be used.

Adhesion molecules play a key role in autoimmune disorders, and serum concentrations of soluble adhesion molecules are increased in Graves' ophthalmopathy (GO). Whether this is due to the strong association with smoking is unknown. It is also not known if the severity or activity of GO determine the serum levels of adhesion molecules. We measured serum concentrations of sICAM-1, sVCAM-1 and sELAM-1 in 62 euthyroid Graves' patients with untreated GO, in 62 healthy controls matched for sex, age and smoking habits, and in 26 euthyroid Graves' patients without GO. GO severity was assessed by the Total Eye Score and the activity by the Clinical Activity Score. Adhesion molecules were measured by highly sensitive ELISAs. GO patients had higher levels than controls (median values in ng/ml with range): sICAM-1 300 [171--575] versus 244 [119--674], P < 0.001; sVCAM-1 457 [317--1060] versus 410 [238--562], P < 0.001; and sELAM-1 61 [19--174] versus 53 [23--118], P = 0.021. Euthyroid Graves' disease patients without GO had levels similar to controls: sICAM-1 273 138--453), sVCAM-1 386 [260--1041] and sELAM-1 46 [22--118]. Smoking had an independent effect and was associated with higher levels of sICAM-1 and lower levels of sVCAM-1 in both GO patients and controls; sELAM-1 levels were comparable. In the 62 GO patients, sICAM-1 correlated significantly with severity of eye disease (r = 0.40, P = 0.002). No correlation was found with the duration of GO, the Clinical Activity Score or TBII levels. Multivariate analysis of all 150 subjects showed that the presence of GO and smoking are independent determinants of sICAM-1 and sVCAM-1 concentrations. In GO patients, the Total Eye Score was a stronger determinant than smoking. It is concluded that (i) smoking is associated with increased sICAM-1 and decreased sVCAM-1 levels; (ii) independent from smoking, euthyroid GO patients have higher levels of sICAM-1, sVCAM-1 and sELAM-1 than patients with euthyroid Graves' disease or healthy controls; (iii) the major determinant of sICAM-1 in GO patients is the severity of their eye disease.

Objective: The Graves' ophthalmopathy quality of life questionnaire (GO-QOL) is the first instrument available to measure health-related quality of life (HRQL) of patients with Graves' ophthalmopathy. The main objective of this study was to define a minimal clinically important difference (MCID) in score on the GO-QOL that can be considered an important improvement in HRQL by examining changes in GO-QOL scores in patients who subjectively report improvement from their treatment. A secondary objective was to test the longitudinal validity of the GO-QOL, using prespecified hypotheses about expected treatment effects., Design: A prospective cohort study., Patients: We included 164 patients who were scheduled for radiotherapy (23), orbital decompression (10 for sight loss, 38 for exophthalmos), eye muscle surgery (31), eyelid lengthening (43) or blepharoplasty (19)., Measurements: Patients completed the GO-QOL and three general HRQL questionnaires, before and three or six months after treatment, depending on the performed procedure. Clinical characteristics were collected from the medical records. Mean changes in GO-QOL scores and effect sizes were calculated after different treatments, and in subgroups of responders and nonresponders according to clinical characteristics and according to the patients themselves., Results: A clinical response to treatment was associated with a change in GO-QOL scores of approximately 10--20 points after major treatments (radiotherapy or decompression), and with a change of approximately 3--10 points after minor surgery (eye muscle surgery, eyelid lengthening, blepharoplasty). Changes in GO-QOL scores of about 6--10 points were considered important improvements by the patients themselves. The direction and amount of change in GO-QOL scores after different treatments were in accordance with our prespecified hypotheses about treatment effects. Effect sizes in the GO-QOL subscales were generally higher than effect sizes of the general HRQL subscales, supporting the longitudinal validity of the GO-QOL., Conclusions: As a general guideline, one could consider a mean change of at least 6 points on one or both subscales an important change in daily functioning for patients. For more invasive therapies, a change of at least 10 points is recommended as a minimal clinically important difference.

Background: Two-thirds of patients with Graves' ophthalmopathy (GO) respond to immunosuppressive treatment (prednisone or orbital irradiation), but one-third do not respond. Responders are likely to be in the active, oedematous stage of the eye disease, nonresponders in the inactive fibrotic stage. Clinical distinction between the two stages can be difficult. Quantitative magnetic resonance imaging (MRI) might be helpful in this respect, as T2 relaxation times are longer in oedematous than in fibrotic tissues., Study Design: Prospective study on quantitative orbital MRI in healthy subjects and patients with moderately severe GO, evaluating T2 relaxation time of extraocular muscles (EOM): (a) in patients vs. controls, and (b) in patients, as a predictor of outcome to orbital irradiation., Subjects and Measurements: Nine healthy volunteers and 64 consecutive patients with moderately severe untreated GO, who were euthyroid for > or = 2 months and qualified for orbital irradiation. T2 relaxation times were measured at the site of the largest diameter in all eight EOM of each subject. Baseline T2 values in patients were related to outcome of radiotherapy, as assessed by an independent observer 6 months later according to predefined criteria., Results: T2 relaxation times of the eight EOM were longer in GO patients than in controls: median [range] 126 [52--250] vs. 88 [50--126] msec; P = 0.003. Per subject the single eye muscle with the longest T2 time was selected to represent active inflammation (excluding the lateral muscles for technical reasons). Again patients were different from controls: 160 [68--250] vs. 103 [86--115] msec, P < 0.001. In the group of 34 responders to subsequent radiotherapy, these T2 times tended to be longer than in the 30 nonresponding patients: 168 [108--250] vs. 138 [68--216] msec, P = 0.07. Using a cut-off value of 130 msec (derived from a receiver-operator-characteristics curve) a positive predictive value of 64% and a negative predictive value of 92% for the outcome of radiotherapy was calculated., Conclusion: Quantitative orbital magnetic resonance imaging was found to be less accurate in predicting successful outcome of radiotherapy than expected. It seems more useful in detecting the fibrotic end-stage than the active stage of Graves' ophthalmopathy. It is therefore of limited use in the decision whether or not to apply immunosuppression in patients with moderately severe Graves' ophthalmopathy.

Increased serum cytokine levels have been reported in patients with autoimmune thyroid disease, but less is known about their levels in patients with Graves' ophthalmopathy (GO). It is not known whether GO is a cell-mediated or humoral autoimmune disease. We investigated whether serum cytokines are elevated in GO patients and whether the cytokines were Th1- or Th2-derived. In addition, elevated cytokines might reflect the activity of GO, and thus we investigated whether cytokine levels could predict the clinical response to orbital radiotherapy. We studied 62 consecutive patients with moderately severe untreated GO and 62 healthy controls, matched for sex, age and smoking habits. Serum concentrations of IL-1RA, sIL-2R, IL-6, sIL-6R, tumour necrosis factor-alpha (TNF-alpha) RI and II and sCD30 were measured using highly sensitive ELISAs, in the patients before and 3 and 6 months after radiotherapy. All patients were euthyroid, with anti-thyroid drugs, before and during the entire study period. All baseline cytokine and cytokine receptor levels were significantly elevated in GO patients compared with healthy controls, except for IL-1RA. The levels did not correlate with parameters of the thyroid disease, nor with the duration, activity or severity of GO. However, backward logistic regression analysis showed that IL-6, sCD30 and TNFalphaRI were able to predict a beneficial response to orbital radiotherapy. We therefore conclude that both Th1- and Th2-derived cytokines are elevated in GO patients compared with its controls. IL-6, sCD30 and TNFalphaRI had some value for predicting therapeutic outcome to orbital irradiation, and may thus reflect active eye disease.

Objective: To study if A-mode ultrasonography (US) in combination with the Clinical Activity Score (CAS) and duration of Graves' ophthalmopathy (GO) could predict the response to immunosuppression in Graves' ophthalmopathy., Design: A prospective clinical study., Patients and Methods: Fifty-six consecutive patients with moderately severe GO were treated with retrobulbar irradiation (10 fractions of 2 Gy). Before treatment the internal reflectivity in the extraocular eye muscles, the CAS, and the duration of Graves' ophthalmopathy were assessed and related to the therapeutic outcome 26 weeks after irradiation., Results: Twenty-eight (50%) of the patients responded favourably to radiotherapy (four patients became worse, and 24 did not change), and the eye muscle echogenecity indeed tended to be lower in the responders than in the non-responders (P = 0.09). From a receiver operator characteristics curve, a cut-off value of 30% yielded a good positive predictive value of 85%, but the negative predictive value was only 60%. The CAS (>/= 4/10) had a positive predictive value of 65%, and a negative predictive value of 56%. When combining the ultrasound with the CAS, the positive predictive value increased to 74% and the negative predictive value to 72%. Adding the duration of eye symptoms (cut-off 18 months), the prediction of response further improved: positive predictive value 79%, and negative predictive value 89%., Conclusions: A-mode ultrasonography has a rather good positive predictive value, but its poor negative predictive value precludes its use as sole activity parameter. By adding the Clinical Activity Score and duration of Graves' ophthalmopathy, the negative predictive value increased considerably. By using this combination, inactive disease can be identified more precise, permitting rehabilitative surgery at an earlier stage in these patients.

Objective: Graves' ophthalmopathy (GO) and Graves' hyperthyroidism are closely associated diseases and thought to be caused by the same autoimmune process. An obvious explanation for this would be the presence of autoantibodies reacting with an autoantigen present in the orbit and the thyroid gland. The TSH-Receptor (TSH-R) antibodies are a likely candidate, because they cause Graves' hyperthyroidism and the TSH-R appears to be present also in orbital tissues. If TSH-R antibodies are responsible for the ophthalmopathy one would expect their titres to correlate with clinical characteristics of the eye disease. The aim of the present study is to see whether TSH-R antibodies are related to the activity and severity of the thyroid-associated ophthalmopathy., Design and Patients: TSH-R antibody levels were measured as TBII (TRAK assay), and TSI (cAMP response of a TSH-R transfected cell line) in serum of 63 patients with untreated moderately severe GO, accompanying Graves' thyroid disease; all patients had been euthyroid for > 2 months., Results: TBII and TSI titres were strongly related to each other. TBII or TSI titres did not correlate with thyroidal or orbital disease duration, nor with TPO antibody levels. In contrast, we found a striking and highly significant correlation between the Clinical Activity Score (CAS) of the eye disease, and both TBII (r = 0.54; P < 0.0001) and TSI (r = 0.50; P < 0.0001). In addition, a weaker but significant relation was found between proptosis (in mm) and TBII (r = 0.36; P = 0.004) and TSI (r = 0.49; P = 0.0001). No correlation was found with eye muscle motility., Conclusion: TSH-R antibody levels correlate directly with clinical features of Graves' ophthalmopathy. The results support the hypothesis of a pathogenetic role of TSH-R antibodies and the TSH-R in the orbit of Graves' ophthalmopathy patients.

To assess the test-retest reliability of a recently developed disease-specific quality of life questionnaire for evaluative studies in patients with Graves' ophthalmopathy (the GO-QOL), measuring visual functioning and psychosocial consequences of changed appearance. Ninety-three patients were included and completed the GO-QOL. Additional information on general quality of life and disease characteristics was obtained. Construct validity and internal consistency of the two subscales was determined, based on principal component analyses, Cronbach's alpha's and correlations with MOS-24, three subscales of the SIP, and clinical measures. Eighty-nine patients completed a second GO-QOL after two weeks including four additional questions about perceived changes in health status. Test-retest reliability was assessed by calculating intraclass correlation coefficients (ICCs) and limits of agreement, using several definitions of stable patients. Slight modifications from the original questionnaire were evaluated for their effect on the validity and reliability. The construct validity of the two subscales was confirmed and Cronbach's alpha's were 0.89 for visual functioning and 0.87 for appearance. The substantial ICCs found for both scales of the GO-QOL (ICCs above 0.80) reflect that the errors of measurement were relatively small, which supports the value of this questionnaire for clinical studies with relatively small sample sizes. The modification of the appearance scale improved the validity of the scale and resulted in less missing values. Following the recommendations of the joint committee of thyroid associations, we recommend the inclusion of HRQL-measures in clinical studies that evaluate treatments for patients with GO. The GO-QOL is a promising tool for this purpose.

Objective: It is thought that immunosuppressive treatment of Graves' ophthalmopathy should be restricted to patients with active eye disease, but assessing disease activity is difficult. Octreotide scintigraphy has been claimed to differentiate active from inactive disease. Here we study the intraobserver variability and diagnostic accuracy of the quantitative measurement of orbital octreotide uptake., Patients and Design: Twenty-two consecutive patients with moderately severe ophthalmopathy were treated with retrobulbar radiotherapy. Pretreatment octreotide scintigraphic data were related to the response at six months after radiotherapy, using Receiving-Operator-Characteristic curves., Measurements: Octreotide uptake was measured at 4 and 24 h after i.v. injection of approximately 3 mCi (= 111 MBq; range 75-150 MBq) 111Indium-DTPA-Octreotide with a neuro-SPECT camera. Counts were measured in fixed regions-of-interest in 4 transversal slices of the orbit, the temporal and the occipital area. Measurements were done twice and intraobserver variability was analysed by coefficients of variations (CV). Uptake is expressed as orbital/background ratio. The nature of the temporal uptake was studied by matching an octreoscan with a technetium scan and MRI., Results: Intra-observer variability of measuring octreotide uptake was acceptable, and the coefficient of variation slightly better using the orbital/occipital ratio (11%), than the orbital/temporal ratio (16%). From matching studies it appears that the temporal uptake takes place, in part, in the parotid gland. The orbital/occipital ratio was used to predict the outcome of radiotherapy. Mean (+/- SD) uptake on the 4 h scan was higher in responders (2.2 +/- 0.66) than in nonresponders (1.7 +/- 0.39; P = 0.04). From the Receiving-Operator-Characteristic curve we determined a cut-off value of 1.85, which yielded a positive predictive value of 92% and a negative predictive value of 70%. The 24 h scan could not predict a response., Conclusion: Quantitative measurement of orbital octreotide uptake is possible. Using the orbital/occipital ratio on the 4 h scan, the octreoscan seems useful in predicting response to subsequent radiotherapy. The 24 h scan seems not to be useful in predicting therapeutic outcome.

There is a need for more reliable and validated methods to assess the eye changes in Graves' disease. Such measurements are now available for the assessment of proptosis, eye muscle dysfunction, and optic nerve involvement. However, no validated objective measurement exists for NO SPECS class II signs. The present study compares the use of colour slides with clinical grading for assessing soft-tissue involvement. Forty-three patients were treated with retrobulbar irradiation. Pre-treatment, and 6 months post-treatment the severity of class II signs was graded from 0 to grade c in two ways; 1) Clinically, by two independent, experienced observers, who recorded their scores on the same day; 2) From colour slides, taken at the same visits, which were graded afterwards in one session independently by the same observers. Inter-observer agreement about clinical grading was low (Kappa 0.32), and was not improved by using the slides (Kappa 0.35). However, by using the clinical scores, the observers disagreed on treatment outcome in 21/43 patients (49%), whereas using the slides disagreement occurred in only 6/43 (14%, p < 0.01). It is concluded that the grading of soft-tissue involvement is highly subjective. However, the use of colour slides does provide a more reliable way to assess a treatment effect and should be used in clinical trials.

Aim: To develop a reliable and valid disease specific quality of life questionnaire (the GO-QOL) for patients with Graves' ophthalmopathy (GO), that can be used to describe the health related quality of life and changes in health related quality of life over time as a consequence of disease and treatment., Methods: 70 consecutive GO patients (age > 18 years) who were referred for the first time to the combined outpatient clinic of the orbital centre and the department of endocrinology completed the 16 questions of the GO-QOL. Additional information on general quality of life and disease characteristics was obtained. Construct validity and internal consistency of the disease specific questionnaire was determined, based on principal component analysis, Cronbach alphas and correlations with MOS-24, three subscales of the SIP, demographic, and clinical measures., Results: The a priori expected subdivision of the questionnaire in two subscales, one measuring the consequences of double vision and decreased visual acuity on visual functioning, and one measuring the psychosocial consequences of a changed appearance, was confirmed in the principal component analysis. Both scales had a good reliability and high face validity. Correlations with other measures supported construct validity. Mean scores (range 0-100) were 54.7 (SD 22.8) for visual functioning and 60.1 (24.8) for appearance (higher score = better health)., Conclusion: The GO-QOL is a promising tool to measure disease specific aspects of quality of life in patients with GO and provides additional information to traditional physiological or biological measures of health status.

To assess the impact of Graves' ophthalmopathy (GO) on quality of life using a general questionnaire, we performed a descriptive study on consecutive ophthalmopathy patients. We included 70 consecutive, euthyroid patients (age >18 years, 50 females, 20 males, mean +/- SD age 53 +/- 13 years) with varying degrees of severity of GO. General quality of life was assessed with the brief survey (24 questions) from the Medical Outcomes Study (MOS-24), and three subscales of the Sickness Impact Profile (SIP). In comparison with a large published reference group, low scores on the MOS-24 were found. Mean +/- SD scores (difference from reference group) were: physical functioning 58 +/- 31 (-28), role functioning 72 +/- 40 (-15), social functioning 78 +/- 25 (-14), mental health 67 +/- 18 (-10), health perceptions 46 +/- 22 (-26), and bodily pain 68 +/- 28 (-6). MOS-24 and SIP scores did not correlate with the duration, severity, or activity of the ophthalmopathy. In conclusion, we have shown that mild to moderately severe GO has a large influence on the quality of life of these patients. The negative impact on well-being seems not to be related to the usual clinical assessment. These results underscore the need for quality-of-life measurements in clinical trials.

Renal biopsies collected from ten patients with serologically confirmed Puumalavirus (PUUV) infection in the acute stage with renal dysfunction who lived in the same PUUV endemic area in The Netherlands, were studied histologically, immuno-histochemically and subsequently examined for the presence of viral antigen by immunoperoxidase staining. Interstitial hemorrhages and deposition of IgM, complement component C3 and fibrin along the basal side of the tubular epithelial cells, were found in PUUV seropositive patients. In six of these patients viral antigen was detected in the cytoplasm of renal tubular epithelial cells with focal distribution in the cortical and medullary areas of the kidneys. Surprisingly, also in two of the ten renal biopsies from PUUV seronegative patients, PUUV antigen was demonstrated, with essentially the same localization. Comparison of severity and extent of the lesions in the seropositive group with those of these two patients showed no significant differences between tubular and interstitial abnormalities. These findings indicate that the demonstration of certain histopathological alterations and the detection of viral antigen by immunoperoxidase techniques in kidney biopsies, may be useful as additional tools for the diagnosis of PUUV infection.

Until 1995, 39 cases of serologically confirmed hantavirusnephropathy in humans, caused by the Puumala type of Hantavirus, have been documented in the Netherlands. Thirty-two of these occurred in Twente, a small region in the eastern part of the country, in which the presence of Puumala-like Hantavirus in feral rodents has recently been demonstrated. Sixteen of the cases documented in humans occurred in 1993. Here we present an overview of clinical, histopathological and epidemiological findings of hantavirus infections. All the clinical and laboratory findings of the 39 documented cases were similar to those found in infections with the same virus in neighbouring countries. Complete recovery of renal function occurred in all patients. One patient developed a Guillain-Barré syndrome after having recovered from her renal failure. Histopathological examination of kidney biopsies collected from 13 of the patients in the acute stage, confirmed the presence of acute interstitial inflammation of differing severity in these individuals. A serological survey carried out amongst 4232 healthy blood donors in the endemic area showed a seroprevalence of 0.7%. This suggests that less severe or perhaps even subclinical infections occur.

Cynomolgus macaques (Macaca fascicularis) were experimentally infected with Puumala virus (strain Hällnäs), which causes nephropathia epidemica in humans in western Europe. During the first week after intratracheal inoculation, the monkeys exhibited signs of lethargy followed by mild proteinuria and microhematuria. Histopathologic changes during the first 7 weeks after infection were largely confined to abnormalities in medullary tubular cells of the kidneys, which coincided with the demonstration of viral antigen and viral RNA. The development of different classes of virus-specific plasma antibodies to the respective viral antigens were similar to those observed in humans with nephropathia epidemica. This first description of a nonhuman primate model for hantavirus infection shows that the cynomolgus macaque provides a suitable model with which to study the pathogenesis of Puumala virus infections and to evaluate new diagnostic methods, immunization strategies, and therapies.

A serological survey for the prevalence of hantavirus infections in The Netherlands was carried out on > 10,000 sera, from selected human populations, and different feral and domestic animal species. Hantavirus-specific antibodies were found in about 1% of patients suspected of acute leptospirosis, 10% of patients with acute nephropathia, and in less than 0.1% haemodialysis and renal transplant patients. Among individuals with a suspected occupational risk, 6% of animal trappers, 4% of forestry workers, 2% of laboratory workers and 0.4% of farmers were seropositive. The majority of the seropositive individuals lived in rural and forested areas. The main animal reservoir of the infection was shown to be the red bank vole (Clethrionomys glareolus). Epidemiological, clinical and laboratory findings seen in serologically confirmed human cases were similar to those associated with nephropathia epidemica.

Sera from Dutch and Belgium individuals who suffered from nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome (HFRS), were tested for the distribution of classes and subclasses of Hantavirus (HV)-specific antibodies at different times after the onset of the disease, with class- and subclass-specific Ig capture enzyme-linked immunosorbent assays (ELISAs). In the acute, early convalescent, and convalescent phases, predominantly specific IgA, IgM, and IgG3 antibodies were detected. Specific IgG2 antibodies were only detected at low levels in the early convalescent and convalescent phases. In the late convalescent phase specific IgG1 and IgG3 antibodies were found, whereas in the late postconvalescent phase only specific IgG1 antibodies proved to be present. Specific IgG4 antibodies were not detected in any of the respective phases. These data show that the simultaneous determination of classes and subclasses of HV specific serum antibodies allows the estimation of the time elapsed after the onset of NE.

We report a patient with myelomonocytic leukaemia, persistent hypokalaemia and lactic acidosis. Cytotoxic chemotherapy induced a short remission with normalisation of the potassium concentration; death resulted from lactic acidosis. These two uncommon complications of leukaemia are discussed briefly.