Search

Background: Primary hyperoxaluria type 1 (PH1) is a genetic disorder resulting in overproduction of hepatic oxalate, potentially leading to recurrent kidney stones, nephrocalcinosis, chronic kidney disease, and kidney failure. Lumasiran, the first RNA interference therapeutic approved for infants and young children, is a liver-directed treatment that reduces hepatic oxalate production. Lumasiran demonstrated sustained efficacy with an acceptable safety profile over 12 months in infants and young children (age <6 years) with PH1 in ILLUMINATE-B (clinicaltrials.gov: NCT03905694), an ongoing, Phase 3, multinational, open-label, single-arm study., Methods: Here, we report interim efficacy and safety findings from ILLUMINATE-B following 30 months of lumasiran treatment. Eligible patients had an estimated glomerular filtration rate (eGFR) >45 ml/min/1.73 m 2 if ≥12 months old or normal serum creatinine if <12 months old, and a urinary oxalate to creatinine ratio (UOx:Cr) greater than the upper limit of normal. All 18 patients enrolled in ILLUMINATE-B completed the 6-month primary analysis period, entered an extension period of up to 54 months, and continue to participate in the study., Results: At Month 30, mean percent change from baseline in spot UOx:Cr was -76%, and mean percent change in plasma oxalate was -42%. eGFR remained stable through Month 30. In 14 patients (86%) with nephrocalcinosis at baseline, nephrocalcinosis grade improved at Month 24 in 12; no patient worsened. In the 4 patients without baseline nephrocalcinosis, nephrocalcinosis was absent at Month 24. Kidney stone event rates were ≤0.25 per person-year through Month 30. Mild, transient injection site reactions were the most common lumasiran-related adverse events (17% of patients)., Conclusion: In infants and young children with PH1, long-term lumasiran treatment resulted in sustained reductions in urinary and plasma oxalate that were sustained for 30 months, with an acceptable safety profile. Kidney function remained stable, low kidney stone event rates were observed through Month 30, and nephrocalcinosis grade improvements were observed through Month 24., Clinical Trial Registration: https://clinicaltrials.gov, identifier NCT03905694., Competing Interests: YF: consultancy fees from Alnylam Pharmaceuticals and membership in the safety review committee. WH: principal investigator for Alnylam Pharmaceuticals; travel and accommodation expenses from Alnylam Pharmaceuticals to attend an international investigators’ meeting. HS-L: principal investigator for Alnylam Pharmaceuticals; travel and accommodation expenses from Alnylam Pharmaceuticals to attend international investigators’ meetings. DJS: grants and other from Alnylam Pharmaceuticals and Dicerna Pharmaceuticals, and personal fees from Advicenne. MM: principal investigator for Alnylam Pharmaceuticals; served on advisory board for Novo Nordisk, Inc. ALS-L: consultancy fees from Alnylam Pharmaceuticals and Dicerna Pharmaceuticals, and principal investigator for research funded by OxThera. JH: consultancy fees from Alnylam Pharmaceuticals. RW and JMG: employees of and shareholders in Alnylam Pharmaceuticals; contributed to study design, data analysis, and (in partnership with all other authors) review and revision of the manuscript in accordance with the ethical principles of Good Publication Practice (GPP 2022) guidelines. DM: research funding, consultancy fees, and non-financial support from Alnylam Pharmaceuticals. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Frishberg, Hayes, Shasha-Lavsky, Sas, Michael, Sellier-Leclerc, Hogan, Willey, Gansner and Magen.)

Introduction: Patients with primary hyperoxaluria type 1 (PH1), a genetic disorder associated with hepatic oxalate overproduction, frequently experience recurrent kidney stones and worsening kidney function. Lumasiran is indicated for the treatment of PH1 to lower urinary and plasma oxalate (POx)., Methods: ILLUMINATE-A (NCT03681184) is a phase III trial in patients aged ≥6 years with PH1 and estimated glomerular filtration rate (eGFR) ≥30 ml/min per 1.73 m 2 . A 6-month double-blind placebo-controlled period is followed by an extension period (≤54 months; all patients receive lumasiran). We report interim data through month 36., Results: Of 39 patients enrolled, 24 of 26 (lumasiran/lumasiran group) and 13 of 13 (placebo/lumasiran group) entered and continue in the extension period. At month 36, in the lumasiran/lumasiran group (36 months of lumasiran treatment) and placebo/lumasiran group (30 months of lumasiran treatment), mean 24-hour urinary oxalate (UOx) reductions from baseline were 63% and 58%, respectively; 76% and 92% of patients reached a 24-hour UOx excretion ≤1.5× the upper limit of normal (ULN). eGFR remained stable. Kidney stone event rates decreased from 2.31 (95% confidence interval: 1.88-2.84) per person-year (PY) during the 12 months before consent to 0.60 (0.46-0.77) per PY during lumasiran treatment. Medullary nephrocalcinosis generally remained stable or improved; approximately one-third of patients (both groups) improved to complete resolution. The most common lumasiran-related adverse events (AEs) were mild, transient injection-site reactions., Conclusion: In patients with PH1, longer-term lumasiran treatment led to sustained reduction in UOx excretion, with an acceptable safety profile and encouraging clinical outcomes.See for Video Abstract., (© 2024 International Society of Nephrology. Published by Elsevier Inc.)

Rationale & Objective: Lumasiran reduces urinary and plasma oxalate (POx) in patients with primary hyperoxaluria type 1 (PH1) and relatively preserved kidney function. ILLUMINATE-C evaluates the efficacy, safety, pharmacokinetics, and pharmacodynamics of lumasiran in patients with PH1 and advanced kidney disease., Study Design: Phase 3, open-label, single-arm trial., Setting & Participants: Multinational study; enrolled patients with PH1 of all ages, estimated glomerular filtration rate ≤45 mL/min/1.73 m 2 (if age ≥12 months) or increased serum creatinine level (if age <12 months), and POx ≥20 μmol/L at screening, including patients with or without systemic oxalosis., Intervention: Lumasiran administered subcutaneously; 3 monthly doses followed by monthly or quarterly weight-based dosing., Outcome: Primary end point: percent change in POx from baseline to month 6 (cohort A; not receiving hemodialysis at enrollment) and percent change in predialysis POx from baseline to month 6 (cohort B; receiving hemodialysis at enrollment). Pharmacodynamic secondary end points: percent change in POx area under the curve between dialysis sessions (cohort B only); absolute change in POx; percent and absolute change in spot urinary oxalate-creatinine ratio; and 24-hour urinary oxalate adjusted for body surface area., Results: All patients (N = 21; 43% female; 76% White) completed the 6-month primary analysis period. Median age at consent was 8 (range, 0-59) years. For the primary end point, least-squares mean reductions in POx were 33.3% (95% CI, -15.2% to 81.8%) in cohort A (n = 6) and 42.4% (95% CI, 34.2%-50.7%) in cohort B (n = 15). Improvements were also observed in all pharmacodynamic secondary end points. Most adverse events were mild or moderate. No patient discontinued treatment or withdrew from the study. The most commonly reported lumasiran-related adverse events were injection-site reactions, all of which were mild and transient., Limitations: Single-arm study without placebo control., Conclusions: Lumasiran resulted in substantial reductions in POx with acceptable safety in patients with PH1 who have advanced kidney disease, supporting its efficacy and safety in this patient population., Funding: Alnylam Pharmaceuticals., Trial Registration: Registered at ClinicalTrials.gov with study number NCT04152200 and at EudraCT with study number 2019-001346-17., Plain-Language Summary: Primary hyperoxaluria type 1 (PH1) is a rare genetic disease characterized by excessive hepatic oxalate production that frequently causes kidney failure. Lumasiran is an RNA interference therapeutic that is administered subcutaneously for the treatment of PH1. Lumasiran has been shown to reduce oxalate levels in the urine and plasma of patients with PH1 who have relatively preserved kidney function. In the ILLUMINATE-C study, the efficacy and safety of lumasiran were evaluated in patients with PH1 and advanced kidney disease, including a cohort of patients undergoing hemodialysis. During the 6-month primary analysis period, lumasiran resulted in substantial reductions in plasma oxalate with acceptable safety in patients with PH1 complicated by advanced kidney disease., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Cixutumumab (CIX) and cetuximab (CET) monoclonal antibodies block ligand-binding to insulin-like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) respectively. The objective of this study was to assess the efficacy of CIX alone or combined with CET in recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) patients., Methods: In this open-label phase II trial, 91 R/M HNSCC patients who progressed within 90 days of platinum-based chemotherapy, were randomized to CIX 10 mg/kg alone or with CET 500 mg/m 2 every 2 weeks. Patients were stratified by prior CET use. The primary endpoint was median progression-free survival (PFS). Exploratory biomarker assessments included relevant markers on archival tumor and serial cytokine/angiogenic-factor profiles in blood., Results: Forty-seven patients were treated with CIX monotherapy and 44 with combination. The median PFS was 1.9 and 2.0 months and clinical benefit rate (complete or partial responses and stable disease) was 5.9% and 15.3%, respectively. There was no exacerbation of CET toxicity by concurrent CIX exposure. Higher tumor expression of IGF-1 was associated with improved PFS in the CIX + CET arm while increased p-EGFR expression correlated with shorter PFS in patients receiving single agent CIX. Higher serum baseline levels of IGF-1 and IGFBP-3 correlated with improved PFS and overall survival (OS) in the CIX arm. Neither regimen resulted in improved PFS or OS compared to historical data with CET alone., Conclusion: The results of this study do not support the use of cixutumumab alone or with cetuximab in unselected patients with R/M HNSCC., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Prader-Willi syndrome (PWS) is a genetic neurodevelopmental disorder of life-threatening hyperphagia, obesity, intellectual deficits, compulsivity, and other behavioral problems. The efficacy and safety of i.n. carbetocin, an oxytocin analog, was evaluated in a prospective, randomized, double-blinded trial in adolescents with PWS., Methods: Eligible patients aged 10-18 years with genetically confirmed PWS were randomized (1:1) to i.n. carbetocin or placebo 3 times daily for 14 days. The primary efficacy endpoint was change in parent/caregiver-rated Hyperphagia in PWS Questionnaire-Responsiveness (HPWSQ-R) total score. Secondary efficacy endpoints included HPWSQ-R behavior, drive, and severity domains; clinician-rated HPWSQ; Children's Yale-Brown Obsessive-Compulsive Severity Scale; food domain of the Reiss Profile; and Clinical Global Impression-Improvement scale. Endpoints were assessed using analysis of covariance. Relationship between primary and secondary endpoints was assessed using Pearson correlation coefficients. Safety was assessed throughout the study., Results: Demographics and clinical characteristics were similar between treatment groups (carbetocin, n = 17; placebo, n = 20). Patients receiving carbetocin had statistically significant reductions in HPWSQ-R total score at study end (-15.6) versus patients receiving placebo (-8.9; P = 0.029); several secondary efficacy endpoints also demonstrated significant differences (P < 0.05). Treatment effects for the primary and secondary endpoints were highly correlated (P ≤ 0.0001). Incidence of adverse events (AEs) was similar between treatment groups., Conclusion: I.n. carbetocin was well tolerated and improved hyperphagia and behavioral symptoms of PWS., Trial Registration: ClinicalTrials.gov: NCT01968187FUNDING. The study was funded by Ferring Pharmaceuticals. Recruitment was aided by ongoing work in PWS performed through Eunice Kennedy Shriver National Institute of Child Health and Human Development grant U54 HD083211.

A simple, sensitive and label-free optical sensor method was developed for allergens analysis using α-casein as the biomarker for cow's milk detection, to be used directly in final rinse samples of cleaning in place systems (CIP) of food manufacturers. A Surface Plasmon Resonance (SPR) sensor chip consisting of four sensing arrays enabling the measurement of samples and control binding events simultaneously on the sensor surface was employed in this work. SPR offers several advantages in terms of label free detection, real time measurements and superior sensitivity when compared to ELISA based techniques. The gold sensor chip was used to immobilise α-casein-polyclonal antibody using EDC/NHS coupling procedure. The performance of the assay and the sensor was first optimised and characterised in pure buffer conditions giving a detection limit of 58ngmL -1 as a direct binding assay. The assay sensitivity can be further improved by using sandwich assay format and amplified with nanoparticles. However, at this stage this is not required as the detection limit achieved exceeded the required allergens detection levels of 2µgmL -1 for α-S1-casein. The sensor demonstrated good selectivity towards the α-casein as the target analyte and adequate recoveries from CIP final rinse wash samples. The sensor would be useful tool for monitoring allergen levels after cleaning procedures, providing additional data that may better inform upon wider food allergen risk management decision(s) that are made by food manufacturer. In particular, this sensor could potentially help validate or optimise cleaning practices for a given food manufacturing process., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Cixutumumab (IMC-A12), a fully human immunoglobulin G1 (IgG1) monoclonal antibody, exerts preclinical activity in several sarcoma models and may be effective for the treatment of these tumours., Methods: In this open-label, multicentre, phase 2 study, patients with previously treated advanced or metastatic rhabdomyosarcoma, leiomyosarcoma, adipocytic sarcoma, synovial sarcoma or Ewing family of tumours received intravenous cixutumumab (10mg/kg) for 1h every other week until disease progression or discontinuation. The primary end-point was the progression-free survival rate (PFR), defined as stable disease or better at 12 weeks. In each tier of disease histology, Simon's optimum 2-stage design was applied (PFR at 12 weeks P0=20%, P1=40%, α=0.10, β=0.10). Stage 1 enrolled 17 patients in each disease group/tier, with at least four patients with stable disease or better required at 12 weeks to proceed to stage 2., Results: A total of 113 patients were enrolled; all tiers except adipocytic sarcoma were closed after stage 1 due to futility. The 12-week PFR was 12% for rhabdomyosarcoma (n=17), 14% for leiomyosarcoma (n=22), 32% for adipocytic sarcoma (n=37), 18% for synovial sarcoma (n=17) and 11% for Ewing family of tumours (n=18). Median progression-free survival (weeks) was 6.1 for rhabdomyosarcoma, 6.0 for leiomyosarcoma, 12.1 for adipocytic sarcoma, 6.4 for synovial sarcoma and 6.4 for Ewing family of tumours. Among all patients, the most frequent treatment-emergent adverse events (AEs) were nausea (26%), fatigue (23%), diarrhoea (23%) and hyperglycaemia (20%)., Conclusions: Patients with adipocytic sarcoma may benefit from treatment with cixutumumab. Cixutumumab treatment was well tolerated, with limited gastrointestinal AEs, fatigue and hyperglycaemia., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Evolution of the env gene in transmitted R5-tropic human immunodeficiency virus type 1 (HIV-1) strains is the most widely accepted mechanism driving coreceptor switching. In some infected individuals, however, a shift in coreceptor utilization can occur as a result of the reemergence of a cotransmitted, but rapidly controlled, X4 virus. The latter possibility was studied by dually infecting rhesus macaques with X4 and R5 chimeric simian simian/human immunodeficiency viruses (SHIVs) and monitoring the replication status of each virus using specific primer pairs. In one of the infected monkeys, both SHIVs were potently suppressed by week 12 postinoculation, but a burst of viremia at week 51 was accompanied by an unrelenting loss of total CD4+ T cells and the development of clinical disease. PCR analyses of plasma viral RNA indicated an env gene segment containing the V3 region from the inoculated X4 SHIV had been transferred into the genetic background of the input R5 SHIV by intergenomic recombination, creating an X4 virus with novel replicative, serological, and pathogenic properties. These results indicate that the effects of retrovirus recombination in vivo can be functionally profound and may even occur when one of the recombination participants is undetectable in the circulation as cell-free virus.

A new pathogenic R5-tropic simian/human immunodeficiency virus (SHIV) was generated following serial passaging in rhesus macaques. All 13 animals inoculated with SHIV(AD8) passaged lineages experienced marked depletions of CD4(+) T cells. Ten of these infected monkeys became normal progressors (NPs) and had gradual losses of both memory and naïve CD4(+) T lymphocytes, generated antiviral CD4(+) and CD8(+) T cell responses, and sustained chronic immune activation while maintaining variable levels of plasma viremia (10(2) to 10(5) RNA copies/ml for up to 3 years postinfection [p.i.]). To date, five NPs developed AIDS associated with opportunistic infections caused by Pneumocystis carinii, Mycobacterium avium, and Campylobacter coli that required euthanasia between weeks 100 and 199 p.i. Three other NPs have experienced marked depletions of circulating CD4(+) T lymphocytes (92 to 154 cells/microl) following 1 to 2 years of infection. When tested for coreceptor usage, the viruses isolated from four NPs at the time of their euthanasia remained R5 tropic. Three of the 13 SHIV(AD8)-inoculated macaques experienced a rapid-progressor syndrome characterized by sustained plasma viremia of >1 x 10(7) RNA copies/ml and rapid irreversible loss of memory CD4(+) T cells that required euthanasia between weeks 19 and 23 postinfection. The sustained viremia, associated depletion of CD4(+) T lymphocytes, and induction of AIDS make the SHIV(AD8) lineage of viruses a potentially valuable reagent for vaccine studies.

We previously reported that passive transfer of polyclonal neutralizing antibodies (NAbs) sufficient to generate a titer of 1:38 in the plasma would confer sterilizing protection to 99% of macaques challenged intravenously with 75 TCID(50) of SHIV(DH12). Neutralizing activity in that study was measured in an MT4 cell assay in which infection was completely blocked (EC(100)). In the current study, the TZM-bl system was used to measure EC(50) neutralizing titers in several of the same macaque plasma samples and the relationship between these titers and in vivo protection was determined. The antiviral EC(50) NAb titers measured in individual plasma samples were higher than those previously obtained in the MT4 system. Furthermore, the geometric mean EC(50) NAb titers against pseudotyped SHIV(DH12) were 33-fold greater than the EC(100) titers measured in the MT4 cell assay against the replication-competent SHIV(DH12) inoculated into animals. An augmented probit regression model was used to generate curves relating TZM-bl EC(50) NAb titers and protection from a virus challenge; estimated titers conferring various levels of protection were then determined. In TZM-bl assays using pseudotyped SHIV(DH12), representative percent in vivo protection/estimated EC(50) titers were 99%/1:4467, 90%/1:1175, 80%/1:676, 50%/1:234, and 33%/1:141. Because it is likely that contributions from other arms of the immune system will contribute to vaccine-induced control, the range of EC(50) NAb titers we have derived may be more informative for evaluating the protective value of NAb activity from TZM-bl assays.

The effect of wet air oxidation on the aerobic biodegradability of a model wastewater containing 1000 mg L(-1) of linear alkylbenzene sulfonate (LAS) has been investigated. Semibatch oxidation experiments were performed temperature of 473 K, oxygen partial pressure of 1.3 MPa and residence times varying from 40 to 390 min, while continuous oxidation experiments were performed at a residence time of 120 min. Oxygen uptake tests were performed to assess the aerobic biodegradability of both the oxidised and the original LAS solutions using cultures that had been adapted to both LAS and oxidation intermediates. The concentration of total organic carbon, chemical oxygen demand and active detergent were followed throughout the wet oxidation and biodegradation experiments, while the main intermediates formed during wet oxidation were identified by means of Electrospray-MS and high performance liquid chromatography. It was found that LAS could be easily oxidised at 473 K to yield a group of molecules with short alkyl chains which do not behave as active detergents. Sulfonated aromatics are produced as intermediates which have had the alkyl chains shortened. The segments of alkyl chains broken off the intermediate compounds appear primarily as short chain organic acids. The original unoxidised 1000 mg L(-1) LAS solution was found to be readily biodegradable in the laboratory aerobic reactors operating at low organic loadings and substrate to microorganism concentration ratios. However, wet oxidation resulted in effluents that were less readily biodegradable than the original LAS with biodegradability decreasing with increasing degree of oxidation. These results suggest that, at the conditions under consideration, a combined chemical pre-oxidations and biological post-treatment process may be less effective in removing LAS than a single-stage biological or chemical process.

Optical monitoring of periodic thin-film stacks by the termination of each layer at the same constant photometric level has certain advantages. One of these principal advantages is the error compensation effect in the vicinity of the monitoring wavelength. In this study, we examine, by simulation, the effect of an error in the knowledge of the absolute value of the photometric termination level on the probable stability in the manufacture of the edge position of a blocked band. The results include equations that allow the determination of the appropriate values of parameters associated with the optimum termination levels to minimize the effects of such errors.

Virus-specific antibodies protect individuals against a wide variety of viral infections. To assess whether human immunodeficiency virus type 1 (HIV-1) envelope-specific antibodies confer resistance against primate lentivirus infections, we purified immunoglobulin (IgG) from chimpanzees infected with several different HIV-1 isolates, and used this for passive immunization of pig-tailed macaques. These monkeys were subsequently challenged intravenously with a chimeric simian-human immunodeficiency virus (SHIV) bearing an envelope glycoprotein derived form HIV-1DH12, a dual-tropic primary virus isolate. Here we show that anti-SHIV neutralizing activity, determined in vitro using an assay measuring loss of infectivity, is the absolute requirement for antibody-mediated protection in vivo. Using an assay that measures 100% neutralization, the titer in plasma for complete protection of the SHIV-challenged macaques was in the range of 1:5-1:8. The HIV-1-specific neutralizing antibodies studied are able to bind to native gp120 present on infectious virus particles. Administration of non-neutralizing anti-HIV IgG neither inhibited nor enhanced a subsequent SHIV infection.

The human immunodeficiency virus type 1 (HIV-1) Vpu and Env proteins are expressed from a bicistronic mRNA. To address the biological significance of the coordinated expression of vpu and env, we compared the relative effects on particle release of HIV-1 isolates containing an intact vpu gene or carrying point mutations in its initiation codon or internal deletions, respectively. We found that the primary AD8 isolate, which is unable to express vpu due to a mutation in its translation initiation codon, was able to replicate in primary macrophages and peripheral blood mononuclear cells with efficiency similar to that of an isogenic variant expressing Vpu. Interestingly, AD8 lacking a vpu initiation codon produced higher levels of Env protein than its Vpu-expressing isogenic variant. In contrast, disabling Vpu without removing the vpu initiation codon did not alter Env expression but significantly reduced virus production. AD8 Env when provided in trans was capable of enhancing release not only of AD8 particles but also of viruses of the T-cell-tropic NL4-3 isolate. We conclude that AD8 Env encodes a Vpu-like activity similar to that previously reported for HIV-2 Env proteins and is thus able to augment virus secretion. When expressed at elevated levels, i.e., following mutation of the vpu initiation codon, AD8 Env was able to compensate for the lack of Vpu and thereby ensure efficient virus release. Thus, the ability to regulate virus release is redundant in AD8 and can be controlled by either Vpu or Env. Since Vpu controls several independent functions, including CD4 degradation, our results suggest that some HIV-1 isolates may have evolved a mechanism to regulate Vpu activity without compromising their ability to efficiently replicate in the host cells.

Background: Despite current recommendations, many people with asthma do not receive annual vaccination against influenza, partly because of concern that vaccine may trigger exacerbations. Colds can trigger exacerbations, which may be mistaken for vaccine-related adverse events. We undertook a double-blind placebo-controlled multicentre crossover study to assess the safety of influenza vaccine in patients with asthma, with allowance for the occurrence of colds., Methods: We studied 262 patients, aged 18-75 years, who recorded daily peak expiratory flow (PEF), respiratory symptoms, medication, medical consultations, and hospital admissions for 2 weeks before the first injection and until 2 weeks after the second injection. Order of injection (vaccine and placebo) was assigned randomly. There was an interval of 2 weeks between injections. The main outcome measure was an exacerbation of asthma within 72 h of injection (defined as a fall in PEF of >20%)., Findings: Among 255 participants with paired data, 11 recorded a fall in PEF of more than 20% after vaccine compared with three after placebo (McNemar's test p=0.06); a fall of more than 30% was recorded by eight after vaccine compared with none after placebo (binomial test p=0.008). However, when participants with colds were excluded, there was no significant difference in the numbers with falls of more than 20% between vaccine and placebo (six vs three; binomial test p=0.51), although the difference for PEF decreases of more than 30% approached significance (five vs none; binomial test, p=0.06). This association was confined to first-time vaccinees., Interpretation: Our findings indicate that pulmonary-function abnormalities may occur as a complication of influenza vaccination. However, the risk of pulmonary complications is very small and outweighed by the benefits of vaccination.

Two primary cell targets for human immunodeficiency virus type 1 (HIV-1) infection in vivo are CD4+ T lymphocytes and monocyte-derived macrophages (MDM). HIV-1 encodes envelope glycoproteins which mediate virus entry into these cells. We have utilized infected and radiolabelled primary peripheral blood mononuclear cell (PBMC) and MDM cultures to examine the biochemical and antigenic properties of the HIV-1 envelope produced in these two cell types. The gp120 produced in MDM migrates as a broad, diffuse band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels compared with that of the more homogeneous gp120 released from PBMCs. Glycosidase analyses indicated that the diffuse appearance of the MDM gp120 is due to the presence of asparagine-linked carbohydrates containing lactosaminoglycans, a modification not observed with the gp120 produced in PBMCs. Neutralization experiments, using isogeneic PBMC and MDM-derived macrophage-tropic HIV-1 isolates, indicate that 8- to 10-fold more neutralizing antibody, directed against the viral envelope, is required to block virus produced from MDM. These results demonstrate that HIV-1 released from infected PBMC and MDM cultures differs in its biochemical and antigenic properties.

Empirically derived formulas are given that allow the thin-film designer to estimate in advance the number of layers needed to meet various thin-film optical performance requirements. The estimation of peak optical density and width of higher-order blocker bands and their suppression are also discussed.

Transgenic mice bearing a murine retroviral envelope transgene (Fv4) have Fv4 gp70env (SU) in their serum in amounts sufficient to block infection by ecotropic virus in vitro. Fv4 Env in serum is derived largely but not exclusively from hematopoietic cells. Tail cells from Fv4 mice and cell lines transduced with the Fv4 env transgene synthesize both components of the envelope protein (gp70 SU and p15E TM) but secrete the gp70 moiety, in the absence of retroviral particles. Blocking of the ecotropic viral receptor by secreted gp70 SU may contribute to resistance to retroviral infection in these mice.

We have used a combination of genetic and immunological techniques to explore how amino acid substitutions in the second conserved (C2) domain of gp120 from human immunodeficiency virus type 1 (HIV-1) affect the conformation of the protein. It was reported previously (R. L. Willey, E. K. Ross, A. J. Buckler-White, T. S. Theodore, and M. A. Martin. J. Viol. 63:3595-3600, 1989) that an asparagine-glutamine (N/Q) substitution at C2 residue 267 of HIV-1 NL4/3 reduced virus infectivity, but that infectivity was restored by a compensatory amino acid change (serine-glutamine; S/N) at residue 128 in the C1 domain. Here we show that the 267 N/Q substitution causes the abnormal exposure of a segment of C1 spanning residues 80 to 120, which compromises the integrity of the CD4-binding site. The reversion substitution at residue 128 restores the normal conformation of the C1 domain and recreates a high-affinity CD4-binding site. The gp120 structural perturbation caused by changes in C2 extends also to the C5 domain, and we show by immunological analysis that there is a close association between areas of the C1 and C5 domains. This association might be important for forming a complex binding site for gp41 (E. Helseth, U. Olshevsky, C. Furman, and J. Sodroski. J. Virol. 65:2119-2123, 1991). Segments of the C1 and C2 domains are predicted to form amphipathic alpha helices. We suggest that these helices might be packed together in the core of the folded gp120 molecule, that the 267 N/Q substitution disrupts this interdomain association, and that the 128 S/N reversion substitution restores it.

The third variable (V3) region within the gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) has been reported to be an important determinant of viral tropism. In this study a series of isogenic recombinant HIV-1 viruses, containing V3 regions from fresh isolates, were examined to ascertain if a relationship exists between viral tropism and specific properties of the virion-associated envelope. All of the viruses were able to infect CD4+ primary lymphocytes, although with different infection kinetics. Several recombinants, however, were unable to infect a continuous CD4+ T-cell line permissive for the parental virus and exhibited a marked decrease in the kinetics of virion-associated gp120 binding to a soluble form of CD4. A known macrophage-tropic HIV-1 isolate, also unable to infect the T-cell line, bound CD4 with similarly slow reaction kinetics. Although the inability to infect T-cell lines is a commonly observed property of macrophage-tropic isolates of HIV-1, the loss of T-cell line tropism by the V3 recombinants was not accompanied by a substantial infectivity for monocyte-derived macrophages, as monitored by reverse transcriptase production. Additional analyses of the recombinant virion gp120s indicated that most of the V3 substitutions increased the inherent stability of the virion gp120-gp41 envelope complex. These results indicate that V3-induced alterations in viral tropism are associated with changes in physical and functional properties of the virion envelope.

Thirteen genetically altered HIV-1 proviruses were created. These various genomes can be segregated into three groups: (i) a set of tat(-) viruses that have a functional HTLV-I Tax inserted in nef; (ii) a set of tat(-) viruses with Gal4 binding sites inserted in U3 and a Gal4-VP16 cDNA inserted in nef; and (iii) a set of tat(+) HIV genomes that are 5' and 3' TAR(-) and are Gal4-binding-site(+) in U3 and Gal4-VP16(+) in nef. We found that viruses in groups (i) and (ii), although tat(-), were fully complemented for viral gene expression based on quantitative measurements of viral protein synthesis and on the visualization by electron microscopy of the proper assembly of morphologically correct virions. Interestingly, group (i) and (ii) virions were defective in a spreading cytopathic infection when assayed in T-lymphocytes. Group (iii) viruses, although capable of producing intact Tat protein, also could not use Tat for transcription/gene expression because of the TAR(-) genotype. However, this class of viral genomes produced viruses that were highly infectious and cytopathic in primary and in continuously propagated T-lymphocytes. These three groups of viruses are all transcriptionally Tat-TAR independent. Their distinct differences in infectivity/cytopathicity provide genetic evidence that Tat provides a transcriptionally independent function in determining infectivity and cytopathicity in the setting of a spreading viral infection. Given that all HIV virions normally contain four intact copies of TAR RNA, our findings suggest a re-examination of whether Tat could be a virion-TAR-associated protein and the possible implications of this for virus infectivity/cytopathicity.

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus receptor for infection of human host cells. We have recently demonstrated that Vpu, a human immunodeficiency virus type 1-encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. Using an in vitro model system, we demonstrated that Vpu targets specific sequences in the cytoplasmic domain of CD4 to promote its degradation. In this report, we have further delineated regions within CD4 which are required for susceptibility to Vpu. Transfer of the CD4 cytoplasmic region into a heterologous protein, CD8, rendered the chimeric protein sensitive to Vpu-dependent degradation. In contrast, substitution of the CD8 transmembrane domain with the analogous region from CD4 did not confer sensitivity to Vpu. Finally, mutant forms of the CD4 protein containing the extracellular region alone or the extracellular and transmembrane regions linked to a heterologous cytoplasmic domain were not targeted by Vpu. Thus, sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to Vpu.

Mutations in the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp120 and gp41, previously shown to confer an enhanced replicative capacity and broadened host range to the ELI1 strain of HIV-1, were analyzed for their biochemical effects on envelope structure and function. The tendency of purified virions to release their extracellular gp120 component, either spontaneously or after interacting with soluble CD4 (CD4-induced shedding) was assessed. A single amino acid substitution in part of the CD4 binding site of gp120 (Gly-427 to Arg) enhanced both spontaneous and CD4-induced shedding of gp120 from virions, while a single change in the fusogenic region of gp41 (Met-7 to Val) affected only CD4-induced shedding. Although each codon change alone conferred increased growth ability, virus with both mutations exhibited the most rapid replication kinetics. In addition, when both of these mutations were present, virions had the highest tendency to shed gp120, both spontaneously and after exposure to soluble CD4. Analysis of CD4 binding to virion-associated gp120 showed that the changes in both gp120 and gp41 contributed to increased binding. These results demonstrated that the increased replicative capacity of the ELI variants in human CD4+ cell lines was associated with altered physical and functional properties of the virion envelope glycoproteins.

The HIV-1 surface glycoprotein gp120 binds CD4 in the initial state of virus-cell fusion. The extensive glycosylation of gp120 has thus far precluded definition of its structure by crystallographic methods. As an initial approach to a gp120 structure, the surface topology was mapped using antibodies. First, the regions of gp120 that are accessible on the surface of the native molecule, and those that are internal but exposed after denaturation, are identified. Second, epitopes for antibodies that recognize complex surface structures comprising segments of different domains are identified. Third, we define how mutations in one domain of gp120 influence the binding of antibodies to defined epitopes on other domains. These latter approaches enable us to start to understand the inter-domain interactions that contribute to the overall structure of the gp120 molecule. Information from these studies is being used to model the structures of individual gp120 domains, and the way in which these interact in the folded protein.

An empirically derived formula, which can be used to predict the average residual reflection that can be expected from an antireflection (AR) coating design as a function of bandwidth, overall thickness, available indices of the coating materials, number of layers, etc., is presented. This formula can be a useful tool not only for the thin-film designer but also for the nondesigner or system engineer to estimate the performance limits of an AR coating for a given application before the design is accomplished. The general predictions are also found to be consistent with the results of two recent AR design competitions involving many independent investigators. Some insight with respect to the basic underlying principles of AR coatings can also be gleaned from the results and the process by which they are found.

Recent findings suggest that a diverse set of interactions exists between crustacean zooplankton and the algae, protozoans and metazoans that live attached to them. The frequent molting of the crustacean exoskeleton keeps these epibiont populations in a state of constant renewal and makes this epibiont community an ideal experimental system for examining the organization of communities whose populations are distributed among ephemeral habitat patches., (Copyright © 1993. Published by Elsevier Ltd.)

The human immunodeficiency virus type 1 Vpu protein is a 16-kDa phosphoprotein which enhances the efficiency of virion production and induces rapid degradation of CD4, the cellular receptor for human immunodeficiency virus. The topology of membrane-inserted Vpu was investigated by using in vitro-synthesized Vpu cotranslationally inserted into canine microsomal membranes. Proteolytic digestion and immunoprecipitation studies revealed that Vpu was a type I integral membrane protein, with the hydrophilic domain projecting from the cytoplasmic membrane face. In addition, several high-molecular-weight proteins containing Vpu were identified by chemical cross-linking. Such complexes also formed when wild-type Vpu and a Tat-Vpu fusion protein were coexpressed. Subsequent analysis by one- and two-dimensional electrophoresis revealed that these high-molecular-weight complexes consisted of homo-oligomers of Vpu. These findings indicate that Vpu is a type I integral membrane protein capable of multimerization.

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus (HIV) receptor for infection of human host cells. We have recently demonstrated that Vpu, an HIV type 1 (HIV-1) encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. In this report, we describe an in vitro model system that allowed us to define important parameters for Vpu-dependent CD4 degradation. The rate of CD4 decay in rabbit reticulocyte lysate was approximately one-third of that observed previously in tissue culture experiments in the presence of Vpu (40 versus 12 min) and required no other HIV-1 encoded proteins. Degradation was contingent on the presence of microsomal membranes in the assay and the coexpression of Vpu and CD4 in the same membrane compartment. By using the in vitro degradation assay, the effects of specific mutations in CD4, including C-terminal truncations and glycosylation mutants, were analyzed. The results of these experiments indicate that Vpu has the capacity to induce degradation of glycosylated as well as nonglycosylated membrane-associated CD4. Truncation of 13 C-terminal amino acids of CD4 did not affect the ability of Vpu to induce its degradation. However, the removal of 32 amino acids from the C-terminus of CD4 completely abolished sensitivity to Vpu. This suggests that Vpu targets specific sequences in the cytoplasmic domain of CD4 to induce its degradation. We also analyzed the effects of mutations in Vpu on its biological activity in the in vitro CD4 degradation assay. The results of these experiments suggest that sequences critical for this function of Vpu are located in its hydrophilic C-terminal domain.

The most common approaches to the application of the Fourier transform method to the synthesis of thin films suffer from two difficulties. The function of reflectance or transmittance to be transformed has been variously approximated, but none of the approximations has been recognized as giving accurate results for all cases. The transformed results for high-reflectance cases have what appear to be distortions in the frequency and in the amplitude scales. These problems and their solutions are the subjects of this paper. It is shown that the reflectance amplitude is the function that should be transformed and that the inclusion of multiple reflections eliminates the distortions in frequency and amplitude.

Many regions within the envelope of human immunodeficiency virus type 1 (HIV-1) that affect its structure and function have been identified. We have previously reported that the interaction of the second conserved (C2) and third variable (V3) regions of gp120 influences the ability of HIV-1 to establish a productive infection in susceptible cells. To better understand the basis for this interaction, we have conducted structure-function analyses of envelope expressed from molecular proviral clones of HIV-1 containing defined mutations in C2 and V3 that individually and in combination differentially affect envelope function. The substitution of a glutamine for an asparagine residue (Q-267) at a potential asparagine-linked glycosylation site in C2, which severely impairs virus infectivity, reduces intracellular processing of gp160 into gp120, the association of gp120 with virions, and the ability of gp120 to bind to the HIV-1 cell surface receptor protein, CD4. The change of an arginine to an isoleucine codon in V3 (I-308), in the presence of the Q-267 mutation, restores virus infectivity to near wild-type levels by increasing the amount of gp120 associated with virions as compared with the Q-267 mutant but does not compensate for the Q-267-induced processing defect. The I-308 change in the context of the wild-type HIV-1 has no affect on processing, association, or CD4 binding. These results indicate that the impaired infectivity of the Q-267 mutant virus is due to a marked reduction in the amount of virion gp120 and suggest that the interaction of C2 and V3 stabilizes the association of gp120 with gp41.

Tissue culture infections of CD4-positive human T cells by human immunodeficiency virus type 1 (HIV-1) proceed in three stages: (i) a period following the initiation of an infection during which no detectable virus is produced; (ii) a phase in which a sharp increase followed by a peak of released progeny virions can be measured; and (iii) a final period when virus production declines. In this study, we have derived equations describing the kinetics of HIV-1 accumulation in cell culture supernatants during multiple rounds of infection. Our analyses indicated that the critical parameter affecting the kinetics of HIV-1 infection is the infection rate constant k = Inn/ti, where n is the number of infectious virions produced by one cell (about 10(2)) and ti is the time required for one complete cycle of virus infection (typically 3 to 4 days). Of particular note was our finding that the infectivity of HIV-1 during cell-to-cell transmission is 10(2) to 10(3) times greater than the infectivity of cell-free virus stocks, the inocula commonly used to initiate tissue culture infections. We also demonstrated that the slow infection kinetics of an HIV-1 tat mutant is not due to a longer replication time but reflects the small number of infectious particles produced per cycle.

We investigated in vivo clot formation on the surface of hydroxyapatite (HA)-coated titanium (Ti) implants and on non-coated Ti implants. Immediately after tooth extraction implant samples were inserted into the blood clot, in the same extraction site, for 1, 30, 60, or 120 seconds. Samples were processed for scanning electron microscopy (SEM). Qualitative observations of clotting topography were made by direct SEM viewing. Neither of the implant surfaces appeared to differ markedly in the degree of clotting during the 120 seconds of implantation; they revealed very early clot formation and limited clot attachment. These results were compared to the findings obtained in a previous study using identical methods with an intact periodontal ligament (PDL), root planed roots, and roots planed and treated with pH 1 citric acid. The PDL surface had the most rapid clot formation at all time periods. By 120 seconds, all root surfaces had completed clot formation.

CD4 is an integral membrane glycoprotein which is known as the human immunodeficiency virus (HIV) receptor for infection of human cells. The protein is synthesized in the endoplasmic reticulum (ER) and subsequently transported to the cell surface via the Golgi complex. HIV infection of CD4+ cells leads to downmodulation of cell surface CD4, due at least in part to the formation of stable intracellular complexes between CD4 and the HIV type 1 (HIV-1) Env precursor polyprotein gp160. This process "traps" both proteins in the ER, leading to reduced surface expression of CD4 and reduced processing of gp160 to gp120 and gp41. We have recently demonstrated that the presence of the HIV-1-encoded integral membrane protein Vpu can reduce the formation of Env-CD4 complexes, resulting in increased gp160 processing and decreased CD4 stability. We have studied the effect of Vpu on CD4 stability and found that Vpu induces rapid degradation of CD4, reducing the half-life of CD4 from 6 h to 12 min. By using a CD4-binding mutant of gp160, we were able to show that this Vpu-induced degradation of CD4 requires retention of CD4 in the ER, which is normally accomplished through its binding to gp160. The involvement of gp160 in the induction of CD4 degradation is restricted to its function as a CD4 trap, since, in the absence of Env, an ER retention mutant of CD4, as well as wild-type CD4 in cultures treated with brefeldin A, a drug that blocks transport of proteins from the ER, is degraded in the presence of Vpu.

An iterative correction process, recently incorporated into the National Research Council of Canada Fourier-transform thin-film synthesis program, is applied to the design of wideband antireflection coatings. This type of problem is different from those solved in the past by this method. It cannot be handled in a practical way without a correction process. We consider in detail the effects-critical for this application-of constraints on the refractive indices and overall thicknesses of the solutions. Our graded-index and multilayer designs have a remarkable resemblance in performance and refractive-index structure to results obtained by more conventional techniques. The Fourier-transform method is of interest because of its speed and versatility.

The mechanisms of human immunodeficiency virus (HIV-1) entry into CD4+ cells and HIV-1 inactivation by sCD4 were studied by analyzing the kinetics of inhibition of viral infection by sCD4 and the kinetics of fusion of CD4+ cells with intact virions labeled with the lipid fluorophore octadecylrhodamine (R18). sCD4 inhibited HIV-1 infection much more effectively when preincubated with virus prior to interaction with CD4+ cells than when mixed simultaneously with virions and cells. The kinetics of inhibition of infection was much slower at 4 degrees and at low sCD4 concentrations than at 37 degrees and at high sCD4 concentrations. In the absence of sCD4, attachment of virus to cells leading to productive infection occurred within 10-30 min. Fusion of the virions with cells started after a 1-2 min lag time and was complete within 15 min. In high-density cell suspensions (5 x 10(7) cells/ml), even very high sCD4 concentrations (100 micrograms/ml) failed to block viral infection during simultaneous mixing of cells, sCD4 and HIV-1. We conclude that the kinetics of sCD4-virus interaction and the competition of sCD4 with the cell surface associated CD4 for the virus are crucial factors in the inhibition of HIV-1 infection by sCD4. These results provide insight into mechanisms of viral penetration into cells and should be considered when designing new approaches for AIDS therapy.

Intracellular transport and processing of the human immunodeficiency virus type 1 (HIV-1) envelope precursor glycoprotein, gp160, proceeds via the endoplasmic reticulum and Golgi complex and involves proteolytic processing of gp160 into the mature virion components, gp120 and gp41. We found that coexpression of gp160 and human CD4 in HeLa cells severely impaired gp120 production due to the formation of intracellular gp160-CD4 complexes. This CD4-mediated inhibition of gp160 processing was alleviated by coexpression of the HIV-1-encoded Vpu protein. The coexpression of Vpu and CD4 in the presence of gp160 resulted in increased degradation of CD4. Although the precise mechanism(s) responsible for the Vpu effect is presently unclear, our findings suggest that Vpu may destabilize intracellular gp160-CD4 complexes.

Intracellular transport and processing of the human immunodeficiency virus type 1 (HIV-1) envelope precursor polyprotein, gp160, proceeds via the endoplasmic reticulum (ER) and Golgi complex. We examined gp160 processing during the production of HIV-1 virions in transfected HeLa cells using wild-type and env mutant proviral molecular clones. Results from pulse-chase analyses indicated that a single amino acid substitution within a highly conserved domain of the env gene impaired gp160 export from the ER, leading to an increase in oligomeric forms of gp160 and a decrease in gp120 production. In contrast, gp160 which contained a mutated cleavage site was able to traverse the ER/Golgi complex, even in the absence of proteolytic processing, and become incorporated into budding virions. These findings indicate that export from the ER is a point in the intracellular trafficking of gp160 that is crucial to the production of the mature envelope components.

This research was designed to evaluate the workplace protection factors of Health and Safety Executive Approved, negative pressure full-facepiece dust respirators, during asbestos stripping. A standard method to measure the concentration of asbestos fibres inside full-facepiece respirators has been developed and the workplace protection factors have been calculated from the data obtained. The respirators in this study are approved for use by the Health and Safety Executive in concentrations of up 900 times the Control Limit [HEALTH AND SAFETY EXECUTIVE, Guidance Note EH 41 (1985)] but the preliminary test results suggest that this level is inappropriately high for this type of respiratory protective equipment.

Uncoated surfaces of high index glasses when cemented to form lens doublets have inferior antireflection properties to doublets of low index glass. This can be overcome by the application of a single layer coating of aluminum oxide prior to cementing.

We report two cases of partial anomalous pulmonary venous drainage in a series of 135 patients with Turner's syndrome.

Double-blind studies of the bronchodilator effects of rimiterol, terbutaline and a combination of rimiterol and terbutaline were performed in patients with chronic asthma. An objective assessment in 21 patients showed no significant differences between the mean improvements in FEV1 during the first 30 minutes after drug administration. At 45 minutes and thereafter terbutaline produced a significantly greater degree of bronchodilatation than rimiterol or the combination of rimiterol and terbutaline. In a subjective study of 27 patients it was found that they were able to detect the more prolonged duration of bronchodilator activity of terbutaline but not the more rapid speed of onset of action of rimiterol.

A previously reported amino acid substitution within the second conserved domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope results in the production of noninfectious particles. Molecular characterization of spontaneous revertant viruses, which arose during long-term cocultures of this env mutant, revealed that an amino acid change within another region of gp120 could functionally compensate for the mutation and restore infectivity. In the current study, we have introduced a conservative amino acid substitution at this second-site revertant codon and observed a marked reduction in HIV-1 infectivity. During the passage of this defective virus in cocultures, yet another revertant appeared which contained an amino acid change within a variable region of gp120 which restored infectivity to near wild-type levels. These results, in combination with other point mutations that have been introduced into the HIV-1 envelope, suggest that at least three discrete regions of gp120 may interact during the establishment of a productive viral infection. This critical step occurs subsequent to the adsorption of virions to the cell surface and either prior to or concomitant with the fusion of viral and cellular membranes.

The platelet attachment and activation potential of a variety of treated, human root surfaces was studied employing an established in vitro method for determining thrombogenicity. In addition, scanning electron microscopic observations were made of the morphological appearance of blood components in initial clot formation on root surfaces containing periodontal ligament (PDL). For the platelet study, the crowns were removed from freshly extracted teeth with and without periodontal disease (PD) and the roots were sectioned. The following surface conditions were created: (1) PDL present, (2) PD, (3) PD planed, (4) PD planed and demineralized, (5) Condition 4 treated with collagenase. All conditions were incubated with Indium-1 ll-labeled platelets with and without the addition of prostacyclin, an inhibitor of platelet activation. It was observed that the greatest number of attached platelets were found in Condition 1, that platelet activation was significantly enhanced in Condition 4 and this effect was reversed by Condition 5. The scanning electron microscopic observations were made on freshly extracted teeth with an intact PDL in which the roots were sectioned and either reinserted immediately into the extraction site or incubated in platelet-rich plasma. It was seen that platelets were involved early in clotting and activated by this surface. This study suggests the possible use of platelet attachment and activation as an indicator for root surface thrombogenicity and perhaps of the fibrous attachment potential.

In a double blind crossover study oral prednisolone was compared with intramuscular depot triamcinolone in the treatment of 20 patients with severe chronic asthma. A short term study comparing each treatment over four weeks showed only minor differences in therapeutic efficacy, but at the end of 24 week periods on each of the two treatments triamcinolone was significantly more effective than prednisolone in terms of forced expiratory volume in one second and forced vital capacity. Better control of asthma was accompanied by a significant fall in weight and some evidence of reduced adrenal suppression (improved cortisol response following a short tetracosactrin test). Side effects, including menstrual irregularities, muscle pain, and hirsuitism, were, however, more common during treatment with triamcinolone.

The efficacy and side-effects of a new corticosteroid, budesonide, was assessed by comparison with beclomethasone dipropionate in double-blind, cross-over study of 30 patients with chronic asthma. The treatment regimens were budesonide 200 micrograms twice daily by conventional pressurized aerosol with a tube spacer attached and beclomethasone 100 micrograms four times daily via a conventional inhaler. Each treatment was given for four weeks. Results were analyzed using Student's t-test for paired comparisons. No significant differences were found for morning and evening peak flow rates, symptom scores, bronchodilator inhaler usage or forced vital capacity. Forced expiratory volume in one second after 4 weeks on each treatment was significantly higher following budesonide therapy (p less than 0.05), but the absolute changes were small and unlikely to be of clinical relevance. There were no major side-effects during either treatment period, but compared with pre-treatment levels serum creatinine and lactic dehydrogenase levels rose significantly during treatment with budesonide (p less than 0.01 and p less than 0.05 respectively). None of these results reflected clinically important biochemical or haematological changes. There was a significant reduction in neutrophil counts following treatment with beclomethasone (p less than 0.05). In the short term treatment of chronic asthma, budesonide administered twice daily is as effective as four times daily beclomethasone. A twice daily dosage regimen should improve patient compliance with therapy.