Yield of Adding chest CT to Abdominal CT to Detect COVID-19 in Patients Presenting With Acute Gastrointestinal Symptoms (SCOUT-3): Multicenter Study.

OBJECTIVE: To determine the incremental yield of standardized addition of chest CT to abdominal CT to detect COVID-19 in patients presenting with primarily acute gastrointestinal symptoms requiring abdominal imaging. Summary Background Data: Around 20% of patients with COVID-19 present with gastrointestinal symptoms. COVID-19 might be neglected in these patients, as the focus could be on finding abdominal pathology. During the COVID-19 pandemic, several centers have routinely added chest CT to abdominal CT to detect possible COVID-19 in patients presenting with gastrointestinal symptoms. However, the incremental yield of this strategy is unknown. METHODS: This multicenter study in 6 Dutch centers included consecutive adult patients presenting with acute nontraumatic gastrointestinal symptoms, who underwent standardized combined abdominal and chest CT between March 15, 2020 and April 30, 2020. All CT scans were read for signs of COVID-19 related pulmonary sequelae using the СО-RADS score. The primary outcome was the yield of high COVID-19 suspicion (СО-RADS 4-5) based on chest CT. RESULTS: A total of 392 patients were included. Radiologic suspicion for COVID-19 (СО-RADS 4-5) was present in 17 (4.3%) patients, eleven of which were diagnosed with COVID-19. Only 5 patients with СО-RADS 4-5 presented without any respiratory symptoms and were diagnosed with COVID-19. No relation with community prevalence could be detected. CONCLUSION: The yield of adding chest CT to abdominal CT to detect COVID-19 in patients presenting with acute gastrointestinal symptoms is extremely low with an additional detection rate of around 1%.

Antigenic variation of foot-and-mouth disease virus serotype A.

The current measures to control foot-and-mouth disease (FMD) include vaccination, movement control and slaughter of infected or susceptible animals. One of the difficulties in controlling FMD by vaccination arises due to the substantial diversity found among the seven serotypes of FMD virus (FMDV) and the strains within these serotypes. Therefore, vaccination using a single vaccine strain may not fully cross-protect against all strains within that serotype, and therefore selection of appropriate vaccines requires serological comparison of the field virus and potential vaccine viruses using relationship coefficients (r1 values). Limitations of this approach are that antigenic relationships among field viruses are not addressed, as comparisons are only with potential vaccine virus. Furthermore, inherent variation among vaccine sera may impair reproducibility of one-way relationship scores. Here, we used antigenic cartography to quantify and visualize the antigenic relationships among FMD serotype A viruses, aiming to improve the understanding of FMDV antigenic evolution and the scope and reliability of vaccine matching. Our results suggest that predicting antigenic difference using genetic sequence alone or by geographical location is not currently reliable. We found co-circulating lineages in one region that were genetically similar but antigenically distinct. Nevertheless, by comparing antigenic distances measured from the antigenic maps with the full capsid (P1) sequence, we identified a specific amino acid substitution associated with an antigenic mismatch among field viruses and a commonly used prototype vaccine strain, A22/IRQ/24/64.

A phase I/II prospective, single arm trial of gefitinib, trastuzumab, and docetaxel in patients with stage IV HER-2 positive metastatic breast cancer.

Inhibition of the HER-2 pathway via the monoclonal antibody trastuzumab has had a major impact in treatment of HER-2 positive breast cancer, but de novo or acquired resistance may reduce its effectiveness. The known interplay between the epidermal growth factor receptor (EGFR) and HER-2 receptors and pathways creates a rationale for combined anti-EGFR and anti-HER-2 therapy in HER-2 positive metastatic breast cancer (MBC), and toxicities associated with the use of multiple chemotherapeutic agents together with biological therapies may also be reduced. We conducted a prospective, single arm, phase I/II trial to determine the efficacy and toxicity of the combination of trastuzumab with the EGFR inhibitor gefitinib and docetaxel, in patients with HER-2 positive MBC. The maximum tolerated dose (MTD) was determined in the phase I portion. The primary end point of the phase II portion was progression-free survival (PFS). Immunohistochemical analysis of biomarker expression of the PKA-related proteins cAMP response element-binding protein (CREB), phospho-CREB and DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa) plus t-DARPP (the truncated isoform of DARPP-32); PTEN; p-p70 S6K; and EGFR was conducted on tissue from metastatic sites. Nine patients were treated in the phase I portion of the study and 22 in the phase II portion. The MTD was gefitinib 250 mg on days 2-14, trastuzumab 6 mg/kg, and docetaxel 60 mg/m(2) every 21 days. For the 29 patients treated at the MTD, median PFS was 12.7 months, with complete and partial response rates of 18 and 46%, and a stable disease rate of 29%. No statistically significant correlation was found between response and expression of any biomarkers. We conclude that the combination of gefitinib, trastuzumab, and docetaxel is feasible and effective. Expression of the biomarkers examined did not predict outcome in this sample of HER-2 overexpressing metastatic breast cancer.

Antigenic and genetic evolution of equine influenza A (H3N8) virus from 1968 to 2007.

Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.

Phase III trial of gemcitabine plus docetaxel versus capecitabine plus docetaxel with planned crossover to the alternate single agent in metastatic breast cancer.

BACKGROUND: Safety and efficacy of gemcitabine plus docetaxel (GD) and capecitabine plus docetaxel (CD) were compared in patients with metastatic breast cancer, where the alternate crossover monotherapy (GD→C or CD→G) was predetermined. PATIENTS AND METHODS: Patients were randomly assigned to 3-week cycles of either gemcitabine 1000 mg/m(2) on days 1 and 8 plus docetaxel 75 mg/m(2) on day 1 or capecitabine 1000 mg/m(2) twice daily on days 1-14 plus docetaxel 75 mg/m(2) day 1. Upon progression, patients received crossover monotherapy. Primary end point was time to progression (TtP). Secondary end points evaluated overall response rate (ORR), overall survival (OS), and adverse events (AEs). RESULTS: Despite over-accrual of 475 patients, the trial matured with only 324 of 385 planned TtP events due to patient discontinuations. Human epidermal growth factor receptor 2 status was not captured in this study. More CD patients (28%) discontinued due to AEs than GD patients (18.0%, P = 0.009). TtP [hazard ratio (HR) = 1.101, 95% confidence interval (CI) 0.885-1.370, P = 0.387] and OS (HR = 1.031, 95% CI 0.830-1.280, P = 0.785) were not significantly different comparing GD and CD. ORR was not statistically different (P = 0.239) comparing GD (72 of 207, 34.8%) and CD (78 of 191, 40.8%). TtP, OS, and ORR were not significantly different comparing crossover groups. GD caused greater fatigue, hepatotoxicity, neutropenia, and thrombocytopenia but not febrile neutropenia; CD caused more hand-foot syndrome, gastrointestinal toxicity, and mucositis. CONCLUSIONS: GD and CD produced similar efficacy and toxicity profiles consistent with prior clinical experience.

Quantifying antigenic relationships among the lyssaviruses.

All lyssaviruses cause fatal encephalitis in mammals. There is sufficient antigenic variation within the genus to cause variable vaccine efficacy, but this variation is difficult to characterize quantitatively: sequence analysis cannot yet provide detailed antigenic information, and antigenic neutralization data have been refractory to high-resolution robust interpretation. Here, we address these issues by using state-of-the-art antigenic analyses to generate a high-resolution antigenic map of a global panel of 25 lyssaviruses. We compared the calculated antigenic distances with viral glycoprotein ectodomain sequence data. Although 67% of antigenic variation was predictable from the glycoprotein amino acid sequence, there are in some cases substantial differences between genetic and antigenic distances, thus highlighting the risk of inferring antigenic relationships solely from sequence data at this time. These differences included epidemiologically important antigenic differences between vaccine strains and wild-type rabies viruses. Further, we quantitatively assessed the antigenic relationships measured by using rabbit, mouse, and human sera, validating the use of nonhuman experimental animals as a model for determining antigenic variation in humans. The use of passive immune globulin is a crucial component of rabies postexposure prophylaxis, and here we also show that it is possible to predict the reactivity of immune globulin against divergent lyssaviruses.

Genetic diversity of equine herpesvirus 1 isolated from neurological, abortigenic and respiratory disease outbreaks.

Equine herpesvirus 1 (EHV-1) causes respiratory disease, abortion, neonatal death and neurological disease in equines and is endemic in most countries. The viral factors that influence EHV-1 disease severity are poorly understood, and this has hampered vaccine development. However, the N752D substitution in the viral DNA polymerase catalytic subunit has been shown statistically to be associated with neurological disease. This has given rise to the term "neuropathic strain," even though strains lacking the polymorphism have been recovered from cases of neurological disease. To broaden understanding of EHV-1 diversity in the field, 78 EHV-1 strains isolated over a period of 35 years were sequenced. The great majority of isolates originated from the United Kingdom and included in the collection were low passage isolates from respiratory, abortigenic and neurological outbreaks. Phylogenetic analysis of regions spanning 80% of the genome showed that up to 13 viral clades have been circulating in the United Kingdom and that most of these are continuing to circulate. Abortion isolates grouped into nine clades, and neurological isolates grouped into five. Most neurological isolates had the N752D substitution, whereas most abortion isolates did not, although three of the neurological isolates from linked outbreaks had a different polymorphism. Finally, bioinformatic analysis suggested that recombination has occurred between EHV-1 clades, between EHV-1 and equine herpesvirus 4, and between EHV-1 and equine herpesvirus 8.

Biochemical and growth inhibition studies of methotrexate and aminopterin analogues containing a tetrazole ring in place of the gamma-carboxyl group.

The biological activities of novel analogues of methotrexate (MTX) and aminopterin (AMT) in which the gamma-carboxyl was replaced by a 1H-tetrazol-5-yl ring, an isosteric group with acidic properties similar to a carboxyl group, were investigated. The tetrazolyl analogues of MTX and AMT were more potent inhibitors of the growth of CCRF-CEM and K562 human leukemia cell lines during continuous (120 h) and 24-h pulse exposure than were the respective parent drugs; only when the exposure time was reduced to 6 h were the parent drugs more potent. These inhibitory effects on growth correlated with the onset of and recovery from inhibition of de novo thymidylate biosynthesis. Growth inhibition by the analogues was protectable by leucovorin. MTX-resistant CCRF-CEM sublines with decreased transport or increased dihydrofolate reductase (DHFR) levels were cross-resistant to the analogues. The analogues were as potent as their parent drugs in inhibiting DHFR activity in vitro and at displacing [3H]MTX from intracellular DHFR. Each analogue was more effective than its parent drug at inhibiting uptake of [3H]MTX into CCRF-CEM cells. The tetrazole analogue of AMT was a linear competitive inhibitor (Kis = 50 microM) of CCRF-CEM folylpolyglutamate synthetase, while the tetrazole analogue of MTX, unlike all other inhibitors, was linear noncompetitive (Kis = 51 microM, Kii = 321 microM). The data suggest that, compared with MTX or AMT, the tetrazole substituent, in place of the gamma-carboxyl group, allows more efficient transport into cells via the reduced folate/MTX carrier and the resulting greater uptake of the analogues leads to inhibition of DNA synthesis and cell death at lower extracellular concentrations during long exposures. The mechanism of cell death could involve inhibition at folypolyglutamate synthetase, but DHFR is the primary target. The low potency of the analogues during short exposure is presumably related to the inability to form the poly-gamma-glutamyl metabolites required for intracellular retention.

Novel 6,5-fused ring heterocyclic antifolates: biochemical and biological characterization.

Six novel antifolates with 2,4-diaminopyrimidine-fused five-membered rings containing either pyrrole or cyclopentene rings were characterized at the cellular and biochemical level. Five of these antifolates were more growth inhibitory to the CCRF-CEM human leukemia cell line than methotrexate [MTX; drug concentration effective at inhibiting cell growth by 50% relative to untreated control (EC50), 12 nM], the antifolate used in the clinic, and two were more potent than 10-ethyl-10-deazaaminopterin (EC50, 2.7 nM); similar patterns of response were obtained in the FaDu and A253 squamous carcinoma cell lines. In addition, the growth inhibitory potency of these antifolates was generally less dependent on exposure time than was MTX. Growth inhibitory effects could be reversed by leucovorin, indicating an antifolate mechanism. These antifolates targeted dihydrofolate reductase (DHFR) based on direct human DHFR inhibition assays [drug concentration inhibiting enzyme activity by 50% (IC50), 0.6-28 nM; MTX IC50, 0.8 nM] and the cross-resistance of MTX-resistant CCRF-CEM cells containing elevated DHFR. Inhibition of human thymidylate synthase was generally weak. These 6,5-fused ring heterocyclic antifolates utilized the reduced folate/MTX transporter for uptake, based on the cross-resistance of MTX uptake-impaired CCRF-CEM cells, and were efficient substrates for this uptake system, based on inhibition of [3H]MTX uptake (IC50, 0.3-5.8 microM; aminopterin IC50, 2.6 microM). These analogues were substrates for CCRF-CEM folylpolyglutamate synthetase, with several being among the most active substrates now known (highest Vrel/Km 0.73; MTX and 10-ethyl-10-deazaaminopterin, 0.013 and 0.24, respectively). Substrate activity for murine intestinal folylpolyglutamate synthetase was also assayed, and a different specificity pattern was observed. These new antifolates are apparently not substrates for aldehyde oxidase. Analogues containing the fused cyclopentene ring are preferred to those containing the fused pyrrole ring based on growth inhibitory potency, effectiveness against decreased uptake mutants and apparent affinity for transport, and inhibition of DHFR. In addition, fused cyclopentene-containing analogues are efficiently polyglutamylated. The data indicate that antifolates with 2,4-diaminopyrimidine-fused five-membered rings, especially those containing the fused cyclopentene ring, are an important new class of antifolates which warrant further exploration at the synthetic and preclinical levels.

Dose ranging phase I study of the serotonin antagonist GR38032F for prevention of cisplatin-induced nausea and vomiting.

GR38032F is a specific 5-HT3 (serotonin) receptor antagonist with antiemetic activity in animal and early human studies. We performed a dose-ranging phase I study of GR38032F in 43 evaluable patients receiving cisplatin 60 120 mg/m2 for the first time (38 of these patients were chemotherapy-naive). Intravenous GR38032F was administered over a dose range from 0.01 to 0.48 mg/kg given three times at four-hour intervals beginning one half hour before cisplatin, and patients were observed for 24 hours. An additional five patients were treated with 0.18 mg/kg given three times at six-hour intervals. Excellent antiemetic efficacy was noted, with 44% of patients experiencing no vomiting and 26% no nausea. Major protection from vomiting (less than or equal to 2 episodes) and from nausea (less than or equal to 2 hours) was experienced by 81% and 44%, respectively. Mild to moderate headache (40%), lightheadedness (21%), and elevated transaminase (19%) were the most common adverse events reported. One patient experienced an apparent hypersensitivity reaction that responded to conventional medications. No extrapyramidal reactions or akathisia were seen. GR38032F was effective through most of the dose range. However, efficacy decreased at the 0.01 mg/kg level and number and intensity of adverse events increased at the 0.48 mg/kg level. Analysis of those patients receiving high-dose cisplatin (100 to 120 mg/m2) revealed a positive association of GR38032F dose and antiemetic activity (Fisher's exact test, two-sided; P less than .05). The 5-HT3 receptor antagonists may provide antiemetic efficacy similar to high-dose metoclopramide without antidopaminergic toxicity. The maximum recommended dose on this schedule of GR38032F is 0.36 mg/kg.

Megestrol acetate and aminoglutethimide/hydrocortisone in sequence or in combination as second-line endocrine therapy of estrogen receptor-positive metastatic breast cancer: a Southwest Oncology Group phase III trial.

PURPOSE: A phase III randomized trial was performed to determine whether combination hormonal therapy with aminoglutethimide (AG) and hydrocortisone (HC) plus megestrol acetate (MA) improved response rates, response duration, or increased survival over the sequential use of each hormone in women with estrogen receptor-positive metastatic breast cancer (MBC) who had maintained stable disease for at least 6 months or responded to tamoxifen. PATIENTS AND METHODS: Two hundred eighty-eight postmenopausal women with progressive estrogen receptor-positive MBC were randomly selected to receive MA 40 mg four times daily (arm I), AG 250 mg four times daily with HC 40 mg daily in divided doses (arm II), versus the combination of MA plus AG given at the same dosages (arm III). Patients on arms I and II who progressed after an adequate trial were crossed over to the other treatment arm. RESULTS: Two hundred thirty-five eligible patients were evaluated for response, time to treatment failure, and survival. Response was only reported for patients with measurable disease and was not statistically different among the three arms. There were two partial responses (PRs) on MA (6%), four complete responses (CRs) and six PRs on AG (24%), and eight PRs and three CRs on MA plus AG (23%) in 32, 42, and 48 measurable patients, respectively. Median times to treatment failure were also similar at 5, 4, and 7 months. Survival was also not statistically different among the three arms at 26, 27, and 26 months for arms I, II, and III, respectively. Toxicity was greater in the two AG arms with respect to fatigue, nausea and vomiting, and rash. CONCLUSION: With the exception of toxicity, there is no response, time to treatment failure, or survival benefit for any one group when comparing MA, AG, or the combination at their stated doses in women with estrogen receptor-positive MBC who had previously responded to or stabilized with tamoxifen.

A human leukemia cell culture system for testing new antifols: differential sensitivity of lymphoid and nonlymphoid cell lines to unconjugated and methotrexate-conjugated polymers of basic amino acids.

A human cell culture system is described for biological testing of potent new folate-targeted antileukemic drugs that are poorly transported. Basic amino acid (lysine and ornithine) polymers were employed as carriers for increasing the uptake of folate analogs by human leukemia cell lines. In growth inhibition assays, the lymphocytic CCRF-CEM line displayed sensitivities to covalent methotrexate (MTX) conjugates of poly-L-lysine (Mr = 15,000, 50,000, or 100,000) or poly-L-ornithine (Mr = 35,000) which were identical to the sensitivities of these cells to the unconjugated polymers during continuous (120 hr) and pulse (24 hr) exposures; both polymers and conjugates were 50-fold less toxic than unconjugated MTX. The growth inhibitory effects of the polymers or MTX-conjugates were not reversed by simultaneous inclusion of leucovorin, while those of MTX were reversed. In contrast, the nonlymphocytic K562 line showed toxicity by the MTX-conjugates at nontoxic levels of the polymers during continuous, but not pulse, exposures. During continuous exposure the conjugates were only 10-fold less toxic than unconjugated MTX. Toxicities of the MTX-conjugates for the K562 line under continuous exposure conditions were reversed by the simultaneous presence of leucovorin or the lysosomotropic agent leupeptin and thus appeared to be a true antifolate effect which required uptake and lysosomal degradation. This human cell line is thus a suitable system in which to study the effects of antifolates which can be coupled to basic polymers.

Cross-resistance studies of folylpolyglutamate synthetase-deficient, methotrexate-resistant CCRF-CEM human leukemia sublines.

CCRF-CEM human leukemia sublines resistant to short-term methotrexate (MTX) exposure as a result of decreased folylpolyglutamate synthetase (FPGS) activity were examined for their response to other cytotoxic agents. The R3/7 and R30dm sublines display 25 and 1%, respectively, of the FPGS activity of CCRF-CEM cells as measured with MTX in vitro. Response to agents in outgrowth experiments was examined under both continuous exposure (120 h, where MTX resistance is not observed) and short-term (6-14.5 h) exposure. During continuous exposure to various classes of agents, cross-resistance of R3/7 and R30dm that correlated with FPGS level was not observed, although some minor (< or = 3-fold) stochastic variations in sensitivity were noted. These agents included actinomycin D, Adriamycin, etoposide, vincristine, cisplatin, cytosine arabinoside, 5-fluorouracil, and some other antifolates. Cross-resistance during continuous exposure that did correlate with FPGS level was noted, however, to glutamate-containing thymidylate synthase inhibitors (including ICI D1694) and, to a minor extent, to 6-mercaptopurine and 5-fluorodeoxyuridine. Slight collateral sensitivity during continuous exposure that apparently correlated with FPGS level was noted to the lipid-soluble antifolate trimetrexate and to 5,8-dideazapteroyl-L-ornithine, an FPGS-specific inhibitor. In short-term exposures (where MTX resistance of the sublines is observed), the resistant sublines displayed sensitivity or cross-resistance to each agent that was qualitatively similar to that observed for the same agent in continuous exposure. Because of the requirement for reduced folates in the anti-DNA mechanism of action of fluoropyrimidines and the current clinical use of leucovorin (LV) to enhance their effects, the interaction of LV and fluoropyrimidines was examined. The results suggest that even highly FPGS-deficient cells are as sensitive to the effects of LV modulation as are wild-type cells even at fluoropyrimidine exposure times as short as 4 h.

Assessing the role of long-distance translocation and spatial heterogeneity in the raccoon rabies epidemic in Connecticut.

Spatial heterogeneity and long-distance translocation (LDT) play important roles in the spatio-temporal dynamics and management of emerging infectious diseases and invasive species. We assessed the influence of LDT events on the invasive spread of raccoon rabies through Connecticut. We identified several putative LDT events, and developed a network-model to evaluate whether they became new foci for epidemic spread. LDT was fairly common, but many of the LDTs were isolated events that did not spread. Two putative LDT events did appear to become nascent foci that affected the epidemic in surrounding townships. In evaluating the role of LDT, we simultaneously revisited the problem of spatial heterogeneity. The spread of raccoon rabies is associated with forest cover--rabies moves up to three-times slower through the most heavily forested townships compared with those with less forestation. Forestation also modified the effect of rivers. In the best overall model, rabies did not cross the river separating townships that were heavily forested, and the spread slowed substantially between townships that were lightly forested. Our results suggest that spatial heterogeneity can be used to enhance the effects of rabies control by focusing vaccine bait distribution along rivers in lightly forested areas. LDT events are a concern, but this analysis suggests that at a local scale they can be isolated and managed.

Synthesis of chelating diamido Sn(IV) compounds from oxidation of Sn(II) and directly from Sn(IV) precursors.

Three dimethyltindiamides containing chelating diamide ligands were synthesised from the reaction of the dilithiated diamine and Me2SnCl2; [SnMe2(L1)] 1 (L1 = κ(2)-N(Dipp)C2H4N(Dipp)), [SnMe2(L2)] 2 (L2 = κ(2)-N(Dipp)C3H6N(Dipp)) and [SnMe2(L3)] 3 (L3 = κ(2)-N(Dipp)SiPh2N(Dipp)), Dipp = 2,6-(i)Pr2C6H3. Reaction of (L2)H2 with SnCl4 and NEt3 led to the formation of the diamidotin dichloride [SnCl2(L2)] 4 whereas reaction of (L1)H2 with SnCl4 and NEt3, or [Sn(L1)] with SnCl4, led to the exclusive formation of the amidotin trichloride [SnCl3{κ(2)-DippN(H)C2H4N(Dipp)}] 5. Reactions of [Sn(L1)] with sulfur and selenium formed [{Sn(L1)(µ-E)}2] (E = S 10 and Se )11. MeI reacted with N-heterocyclic stannylenes to generate the Sn(iv) addition products [Sn(Me)I(L1)] 12, [Sn(Me)I(L2)] 13, [Sn(Me)I(L3)] 14 and [Sn(Me)I(L4)] 15 (L4 = κ(3)-N(Dipp)C2H4OC2H4N(Dipp)), and subsequent reaction with AgOTf (OTf = OSO2CF3) generated the corresponding Sn(iv) triflates [Sn(Me)OTf(L1)] 16, [Sn(Me)OTf(L2)] 17 and [Sn(Me)OTf(L4)] 19 with [Sn(Me)OTf(L3)] 18 formed only as a mixture with unidentified by-products. All of the compounds were characterised by single crystal X-ray diffraction.

The view of European experts regarding health economics for medical nutrition in disease-related malnutrition.

Health-care systems are currently facing tremendous budget constraints resulting in growing pressure on decision makers and health-care providers to obtain the maximum possible health benefits of the resources available. Choices have to be made, and health economics can help in allocating limited health-care resources among unlimited wants and needs. Attempts to achieve cost reductions often focus on severe pathologies and chronic diseases as they commonly represent high health-care expenditures. In this context, awareness of the considerable financial burden caused by disease-related malnutrition (DRM) is lacking. Possibilities of reducing costs by optimising the management of DRM through medical nutrition will mostly not even be taken into account. During a European expert meeting, the total evaluation of medical nutrition was viewed and discussed. The aim of this meeting was to gain an experts' outline of the key issues relating to the health economic assessment of the use of medical nutrition. This article provides a summary of the observations per discussed item and describes the next steps suggested.

Optimization and comparison of the MTT assay and the 3H-TdR assay for the detection of IL-2 in helper T cell precursor assays.

The helper T cell precursor (HTLp) assay is of value for predicting graft-versus-host disease after allogeneic bone marrow transplantation. The assay requires reliable detection of the amount of interleukin 2 (IL-2) produced by one cell. To optimize the IL-2 sensitivity of our HTLp assay we tested an IL-2 dependent cell line, CTLL-2, with two different measurement methods: a colorimetric assay with tetrazolium (MTT) and an isotope incorporation assay with 3H-thymidine (3H-TdR). The test conditions examined encompassed: time without IL-2, preincubation time in IL-2, CTLL-2 cell concentration and different human sera. Due to the different measurement procedures, the volumes of the IL-2 dilutions were 75 microl in assays with MTT and 150 microl in assays with 3H-TdR. We found that it was the amount of IL-2, not the concentration, that limited the growth of CTLL-2 cells. In the most optimal setting the MTT assay could detect 0.6 pg IL-2/well, corresponding to 8 pg/ml. For the 3H-TdR assay the sensitivity was 0.6 pg/well, corresponding to 4 pg/ml. Because of the possibility of IL-2 detection in the whole culture volume (150 microl), we found that the 3H-TdR assay was superior to the MTT assay with a 10-fold better sensitivity in different human sera.

Design of limiting dilution analysis experiments for helper T lymphocyte precursor frequency determination in the context of allogeneic bone marrow transplantation.

Helper, interleukin 2 (IL-2) producing, T lymphocyte precursor (HTLp) frequency determination by limiting dilution analysis (LDA) is of value for quantifying alloreactivity in allogeneic bone marrow transplantation (BMT). LDA assays are labour-intensive and time-consuming to perform and the numbers of donor and recipient cells available are limited. It is therefore important that the design of the experiment yields reliable frequencies with a minimum of effort and a realistic cell requirement. We have critically evaluated the methods proposed for LDA design by Strijbosch et al. [Strijbosch, L.W., Buurman, W.A., Does, R.J., Zinken, P.H., Groenewegen, G., 1987. Limiting dilution assays. Experimental design and statistical analysis. J. Immunol. Methods 97, 133] and by Blackett and Gordon [Blackett, N.M., Gordon, M.Y., 1996. Optimizing limiting dilution assays: frequency and 'ability' measurements of haemopoietic progenitor cells. Br. J. Haematol. 92, 507 (see comments)] and found them inadequate for this application. The estimation of the HTLp frequency is traditionally based on the single-hit Poisson model and the adequacy of this model was compared with that of a double-hit model. The results were in favour of the single-hit model. Ten different LDA experimental designs were explored by Monte Carlo simulations. The optimal design exploits the maximal numbers of cells that can be obtained for analysis to estimate HTLp frequencies in the range 1:1,000,000-1:20,000 with a coefficient of variation of 10-20% and with a minimum of manual labour.

Autoreactivity, backstimulation and reproducibility in a helper T lymphocyte precursor assay.

Helper T lymphocyte precursor (HTLp) frequencies determined by limiting dilution analysis (LDA) have a predictive value for alloreactivity in allogeneic bone marrow transplantation. Methodological problems in LDA include autoreactivity in the responder or stimulator cell populations and interleukin 2 (IL-2) production by the stimulator cells as a response to the responder cells (backstimulation). The extent and impact of these aspects for IL-2 production and HTLp frequency determination were studied by autologous and allogeneic mixed lymphocyte reactions with healthy volunteers and HTLp determinations from bone marrow transplantation donor/recipient pairs. We found that autoreactivity occurred in the unirradiated cells with a reproducible inter-individual variation. The immunogenicity of the stimulator cells was preserved after gamma irradiation with 50 Gy and the risks of autoreactivity and backstimulation were limited. Higher doses of irradiation decreased the immunogenicity. Immune reactions to antigens present in the serum supplement of the culture medium were seen with foetal calf serum and to a lesser extent with pooled human sera. This could be avoided by the use of autologous serum. We were unable to ensure satisfactory culture conditions in serum-free medium. The reproducibility of the HTLp frequency determinations was tested for intra- and inter-assay variation. The coefficients of variation were estimated as 24% and 35%, respectively. This was acceptable considering the range of the HTLp frequencies (1:10(2) to 1:10(7)). The influence of the extent of autoreactivity of the bone marrow donors was investigated in 28 HLA-identical sibling transplantations. We found no correlation between the autoreactivity of the donors and the HTLp frequencies. The extent of autoreactivity of the donor did not correlate with the clinical outcome in terms of acute graft-versus-host disease, treatment-related mortality, risk of relapse and overall survival. In spite of methodological difficulties and interference from autoreactivity and backstimulation, reproducible quantification of clinically significant alloreactivity can be attained.

Evaluation of X-radiation and UV-B radiation for elimination of 3H-thymidine incorporation into responder cells in the IL-2 producing helper T lymphocyte precursor assay.

The helper T lymphocyte precursor (HTLp) assay is of value for predicting graft-vs.-host disease after allogeneic bone marrow transplantation. The assay is based on limiting dilution analysis and requires detection of the amount of interleukin 2 (IL-2) produced by one activated T cell. IL-2 is detected by 3H-thymidine (3H-TdR) incorporation into the IL-2 dependent cell line, CTLL-2. Detection of IL-2 in the whole culture volume of the wells in the microtiter plates increases sensitivity, but requires elimination of 3H-TdR incorporation into the responder cells in the HTLp assay. We have compared the ability of X-radiation and ultraviolet B (UV-B) radiation to eliminate 3H-TdR incorporation. X-irradiation of the cells reduced 3H-TdR incorporation by 90% with doses up to 400 Gy, but the incorporation was still 10 times higher than in wells with stimulator cells only. UV-B irradiation (with Philips TL 12 tubes) of the cells with > or = 2 J/cm2 decreased 3H-TdR incorporation to the level of wells with stimulator cells only. Neither X-irradiation nor UV-B irradiation of the cultures affected IL-2 produced in the assay or human recombinant IL-2 measured by 3H-TdR incorporation into the IL-2 dependent cell line, CTLL-2. HTLp frequencies determined after 25 Gy X-irradiation were higher (mean 1.5, range 1.02-2.2 times higher) than HTLp frequencies determined after UV-B irradiation with 2 J/cm2.