Interferon-Induced Transmembrane Protein 1 Restricts Replication of Viruses That Enter Cells via the Plasma Membrane.

The acute antiviral response is mediated by a family of interferon-stimulated genes (ISGs), providing cell-intrinsic immunity. Mutations in genes encoding these proteins are often associated with increased susceptibility to viral infections. One family of ISGs with antiviral function is the interferon-inducible transmembrane proteins (IFITMs), of which IFITM3 has been studied extensively. In contrast, IFITM1 has not been studied in detail. Since IFITM1 can localize to the plasma membrane, we investigated its function with a range of enveloped viruses thought to infect cells by fusion with the plasma membrane. Overexpression of IFITM1 prevented infection by a number of Paramyxoviridae and Pneumoviridae, including respiratory syncytial virus (RSV), mumps virus, and human metapneumovirus (HMPV). IFITM1 also restricted infection with an enveloped DNA virus that can enter via the plasma membrane, herpes simplex virus 1 (HSV-1). To test the importance of plasma membrane localization for IFITM1 function, we identified blocks of amino acids in the conserved intracellular loop (CIL) domain that altered the subcellular localization of the protein and reduced antiviral activity. By screening reported data sets, 12 rare nonsynonymous single nucleotide polymorphisms (SNPs) were identified in human IFITM1, some of which are in the CIL domain. Using an Ifitm1-/- mouse, we show that RSV infection was more severe, thereby extending the range of viruses restricted in vivo by IFITM proteins and suggesting overall that IFITM1 is broadly antiviral and that this antiviral function is associated with cell surface localization.IMPORTANCE Host susceptibility to viral infection is multifactorial, but early control of viruses not previously encountered is predominantly mediated by the interferon-stimulated gene (ISG) family. There are upwards of 300 of these genes, the majority of which do not have a clearly defined function or mechanism of action. The cellular location of these proteins may have an important effect on their function. One ISG located at the plasma membrane is interferon-inducible transmembrane protein 1 (IFITM1). Here we demonstrate that IFITM1 can inhibit infection with a range of viruses that enter via the plasma membrane. Mutant IFITM1 proteins that were unable to localize to the plasma membrane did not restrict viral infection. We also observed for the first time that IFITM1 plays a role in vivo, and Ifitm1-/- mice were more susceptible to viral lung infection. These data contribute to our understanding of how ISGs prevent viral infections.

A decision impact, decision conflict and economic assessment of routine Oncotype DX testing of 146 women with node-negative or pNImi, ER-positive breast cancer in the U.K.

BACKGROUND: Tumour gene expression analysis is useful in predicting adjuvant chemotherapy benefit in early breast cancer patients. This study aims to examine the implications of routine Oncotype DX testing in the U.K. METHODS: Women with oestrogen receptor positive (ER+), pNO or pN1mi breast cancer were assessed for adjuvant chemotherapy and subsequently offered Oncotype DX testing, with changes in chemotherapy decisions recorded. A subset of patients completed questionnaires about their uncertainties regarding chemotherapy decisions pre- and post-testing. All patients were asked to complete a diary of medical interactions over the next 6 months, from which economic data were extracted to model the cost-effectiveness of testing. RESULTS: Oncotype DX testing resulted in changes in chemotherapy decisions in 38 of 142 (26.8%) women, with 26 of 57 (45.6%) spared chemotherapy and 12 of 85 (14.1%) requiring chemotherapy when not initially recommended (9.9% reduction overall). Decision conflict analysis showed that Oncotype DX testing increased patients' confidence in treatment decision making. Economic analysis showed that routine Oncotype DX testing costs £6232 per quality-adjusted life year gained. CONCLUSION: Oncotype DX decreased chemotherapy use and increased confidence in treatment decision making in patients with ER+ early-stage breast cancer. Based on these findings, Oncotype DX is cost-effective in the UK setting.

Impact and outcomes after lymphaticovenous anastomosis for 150 cases of lymphoedema followed up over 24 months.

BACKGROUND: Lymphoedema is a chronic condition affecting daily activities of life, causing significant alterations and adjustments. Since 2015, lymphaticovenous anastomosis (LVA) has been available on the National Health Service for people with lymphoedema in Wales, United Kingdom. This study aimed to explore the impact and outcomes after LVA over a 24-month follow-up. METHODS: Data were prospectively captured before and after LVA surgery on 150 patients with unilateral upper or lower limb lymphoedema. The same team (three lymphoedema specialists and two plastic surgeons) assessed/operated on all patients. Data captured included a quality of life tool (EQ5D5L), circumferential measurements (tape measure/perometer), compression garment usage, occurrence of cellulitis and a range of patient-reported outcome measures. RESULTS: People who underwent LVA surgery had predominantly cancer-related lymphoedema (n = 118). Reviewing baseline data and 24-months after LVA, quality of life statistically improved (p = <0.005), as well as pain, heaviness, anxiety, impact on hobbies, work, purchasing clothes and intimacy/desirability. Mean perometer and circumferential measurements did not reduce over the 24 months. Number of days per week and hours that the patient wore compression garments did lessen and was statistically significant (p = <0.001). The quantity of cellulitis episodes captured from two years before and two years after LVA decreased from 4.22 to 0.10 (p = <0.001). Significant results (p = <0.001) were also found in a reduction in patients taking days off work due to cellulitis (5.81 to 0.16). CONCLUSION: LVA resulted in significant improvements in patient-reported outcome measures, cellulitis episodes and reduced need for compression garments. Limb circumference via tape measure/perometer did not alter, yet the patient's quality of life considerably improved.

A study protocol for a double-blind randomised placebo-controlled trial evaluating the efficacy of carrageenan nasal and throat spray for COVID-19 prophylaxis-ICE-COVID.

INTRODUCTION: At present, vaccines form the only mode of prophylaxis against COVID-19. The time needed to achieve mass global vaccination and the emergence of new variants warrants continued research into other COVID-19 prevention strategies. The severity of COVID-19 infection is thought to be associated with the initial viral load, and for infection to occur, viruses including SARS-CoV-2 must first penetrate the respiratory mucus and attach to the host cell surface receptors. Carrageenan, a sulphated polysaccharide extracted from red edible seaweed, has shown efficacy against a wide range of viruses in clinical trials through the prevention of viral entry into respiratory host cells. Carrageenan has also demonstrated in vitro activity against SARS-CoV-2. METHODS AND ANALYSIS: A single-centre, randomised, double-blinded, placebo-controlled phase III trial was designed. Participants randomised in a 1:1 allocation to either the treatment arm, verum Coldamaris plus (1.2 mg iota-carrageenan (Carragelose®), 0.4 mg kappa-carrageenan, 0.5% sodium chloride and purified water), or placebo arm, Coldamaris sine (0.5% sodium chloride) spray applied daily to their nose and throat for 8 weeks, while completing a daily symptom tracker questionnaire for a total of 10 weeks. PRIMARY OUTCOME: Acquisition of COVID-19 infection as confirmed by a positive PCR swab taken at symptom onset or seroconversion during the study. Secondary outcomes include symptom type, severity and duration, subsequent familial/household COVID-19 infection and infection with non-COVID-19 upper respiratory tract infections. A within-trial economic evaluation will be undertaken, with effects expressed as quality-adjusted life years. DISCUSSION: This is a single-centre, phase III, double-blind, randomised placebo-controlled clinical trial to assess whether carrageenan nasal and throat spray reduces the risk of development and severity of COVID-19. If proven effective, the self-administered prophylactic spray would have wider utility for key workers and the general population. TRIAL REGISTRATION: NCT04590365; ClinicalTrials.gov NCT04590365. Registered on 19 October 2020.

IFITM3 restricts virus-induced inflammatory cytokine production by limiting Nogo-B mediated TLR responses.

Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor that limits viral pathogenesis and exerts poorly understood immunoregulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 promotes MyD88-dependent, TLR-mediated IL-6 production following exposure to cytomegalovirus (CMV). IFITM3 also restricts IL-6 production in response to influenza and SARS-CoV-2. In dendritic cells, IFITM3 binds to the reticulon 4 isoform Nogo-B and promotes its proteasomal degradation. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesis in vivo, and in the case of TLR2 responses, this process involves alteration of TLR2 cellular localization. Nogo-B deletion abrogates inflammatory cytokine responses and associated disease in virus-infected IFITM3-deficient mice. Thus, we uncover Nogo-B as a driver of viral pathogenesis and highlight an immunoregulatory pathway in which IFITM3 fine-tunes the responsiveness of myeloid cells to viral stimulation.

Anti-HLA donor-specific antibodies detected in positive B-cell crossmatches by Luminex predict late graft loss.

The significance of B-cell crossmatching in kidney transplantation is controversial. Recipients (n = 471) transplanted in a single centre from 1987 to 2005 with complete T- and B-cell crossmatch records were studied. Sera from 83 patients transplanted across a positive B-cell crossmatch, with concomitant negative T-cell crossmatch (T-B+) on either current and/or peak sera were studied using Luminex to determine presence of donor-specific antibodies (DSA). Clinical outcomes of T-B+ patients were compared with 386 T-B- patients. T-B+ predicted vascular (p = 0.01), but not cellular (p = 0.82) or glomerular (p = 0.14) rejection. IgG HLA DSA were found in 33% (n = 27) of the T-B+ patients and were associated with higher risk of any (p = 0.047), vascular (p = 0.01) or glomerular (p < 0.001) rejection at 6 months. Of 27 patients with DSA, 18/21 (86%) were the complement-fixing IgG(1) and/or IgG(3) subclass antibodies. DSA imposed a statistically significant higher risk of graft loss 5 years posttransplant (1.8 [1.0-3.3], p = 0.045). This study showed that only one-third of positive B-cell crossmatch (BXM) was caused by DSA and was associated with late graft loss. Thus, using BXM to preclude kidney transplantation may potentially disadvantage >60% of patients in whom BXM is not indicative of the presence of DSA.

Fissure Seal or Fluoride Varnish? A Randomized Trial of Relative Effectiveness.

Fissure sealant (FS) and fluoride varnish (FV) are effective in preventing dental caries when compared with a no-treatment control. However, the relative clinical effectiveness of these interventions is uncertain. The objective of the study was to compare the clinical effectiveness of FS and FV in preventing dental caries in first permanent molars (FPMs) in 6- to 7-y-olds. The study design was a randomized clinical trial, with 2 parallel arms. The setting was a targeted-population program that used mobile dental clinics in schools located within areas of high social and economic deprivation in South Wales. A total of 1,016 children were randomized 1:1 to receive either FS or FV. Resin-based FS was applied to caries-free FPMs and maintained at 6-mo intervals. FV was applied at baseline and at 6-mo intervals for 3 y. The main outcome measures were the proportion of children developing caries into dentine (D4-6MFT) on any 1 of up to 4 treated FPMs after 36 mo. At 36 mo, 835 (82%) children remained: 417 in the FS arm and 418 in the FV arm. A smaller proportion of children who received FV ( n = 73, 17.5%) versus FS ( n = 82, 19.6%) developed caries into dentine on at least 1 FPM (odds ratio [OR] = 0.84; 95% CI, 0.59 to 1.21; P = 0.35), a nonstatistically significant difference between FS and FV treatments. The results were similar when the number of newly decayed teeth (OR = 0.86; 95% CI, 0.60 to 1.22) and tooth surfaces (OR = 0.85; 95% CI, 0.59 to 1.21) were examined. In a community oral health program, semiannual application of FV resulted in caries prevention that was not significantly different from that obtained by applying and maintaining FS after 36 mo (EudraCT: 2010-023476-23; ISRCTN: ISRCTN17029222).

Evolution in HLA-DRB1 and major histocompatibility complex class II haplotypes of Australian aborigines. Definition of a new DRB1 allele and distribution of DRB1 gene frequencies.

The distribution of HLA-DRB1 alleles was studied in Australian aborigines from different parts of Australia. There were significant differences in the frequencies of DRB1*0412, 1409, and 1410 between the Central Desert and Yuendumu populations and the previously reported Cape York and Kimberley aboriginal populations. A new DRB1 allele, DRB1*1414, present at low frequency in the Central Desert population, was identified. DRB1*1414 appears to be closely related to DRB1*1407 and is proposed to have arisen by intragenic recombination. A novel DR-DQ haplotype, DRB1*1402-DRB3*0101-DQA1*0501-DQB1*0402, was also identified. This haplotype may be ancestral to the DRB1*1409-DQB1*0402 haplotype present in these populations. The presence of alleles and haplotypes apparently confined to Australian aboriginal populations and differences in the distribution of these alleles in different populations suggests that evolution has occurred in the class II region in the period since colonization of Australia, an estimated 50,000 years ago.

Genetic susceptibility and the link between cat exposure and rheumatoid arthritis.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic inflammatory disease for which immunogenetic susceptibility factors have been defined. In a recent case control study, it was shown that a prior intimate relationship with pet cats or budgerigars confers risk for subsequent development of RA after a period of latency. Pets are a potential reservoir for putative microbial agents that could be a stimulus for chronic inflammation subject to the influence of immunogenetic factors. Therefore, a study was undertaken to determine whether the presence of HLA-DRB1 alleles bearing the RA susceptibility motif influenced risk for RA associated with prior exposure to pets. METHODS: Blood samples were obtained from available RA patients and their case controls who had participated in the prior epidemiologic study. DR and DQ genotypes were determined by sequence analysis of oligonucleotides amplified from the DRB1 and DQB1 genes by polymerase chain reactions (PCR). Subjects were segregated according to pet exposure (as determined previously) and genotype for statistical analyses. RESULTS: The odds ratio (OR) for prepubertal exposure to cats and RA in available subjects irrespective of DRB1 genotype was 4.2 (CI, 2.1 to 8.5; P<.00002). The OR between prior exposure to cats and RA in subjects with the RA susceptibility genotype DRB1 *0401 and *0404 was 5.8 (CI, 1.4 to 26; P<.02) and >24 (CI, 1.6 to 813; P<.01), respectively. In subjects with the genotype DRB1 *1501, the association between RA and prior cat exposure was OR 8.4 (CI, 1.7 to 45; P<.01). No significant association between RA and pet exposure was found in patients selected according to other genotypes. The association between RA and the recognized HLA-DR susceptibility motif was slightly stronger in subjects with a history of intimate cat exposure (OR 4.7 [CI, 1.5 to 14.8], P<.005) than subjects without prior intimate exposure (OR 3.3 [CI; 1.2 to 9.3], P<.02). In the small number of subjects who had reported an intimate association with pet birds, no influence of DR genotype on risk for RA was discerned. CONCLUSIONS: Risk for RA associated with prior intimate exposure to cats is concentrated in subjects with the RA-susceptibility conferring genotypes DRB1 *0401 and *0404. The findings suggest an interaction between an environmen-tal agent associated with pet cats and certain RA susceptibility-conferring DR genotypes. The risk for RA associated with intimate cat exposure also was significant in subjects with DRB1*1501, a genotype not otherwise associated with RA, but which shares with known RA susceptibility-bearing alleles the presence of an electropositive pocket (Pocket 4) in the DR peptide binding groove.

The common marmoset as a novel preclinical transplant model: identification of new MHC class II DRB alleles and prediction of in vitro alloreactivity.

The difficulties with using nonhuman primate species such as rhesus macaques and baboons have led us to investigate the common marmoset (Callithrix jacchus) as an alternative preclinical model for transplantation research. This requires reliable methods of detecting alloreactivity between donor and recipient pairs, particularly if colonies are inbred and share just a few common alleles for leucocyte antigens. We firstly identified marmoset major histocompatibility complex (MHC) Class II DRB genes (Caja-DRB*W1201, Caja-DRB1*03, Caja-DRB*W16) using sequence-based typing techniques. Genomic DNA (n= 49) was extracted from whole blood or spleen tissue. Exon 2 of target genes was amplified by PCR using primers specific for known marmoset alleles, and then sequenced using ABI PRISM((R)) Big Dye Terminator technology and Assign sequence analysis software. DRB*W1201 was universally present. Eight DRB*W16 alleles and five DRB1*03 alleles were identified in this colony. We also identified two previously unreported DRB*W16 alleles, and confirmed inheritance of these alleles within several sibling groups. Subsequently, we investigated whether matching at MHC Class II DRB loci alone could predict alloreactivity, as assessed in vitro by two-way mixed lymphocyte reactions (MLRs). Fully DRB-matched, partially mismatched and fully mismatched animal pairs were prospectively chosen. MLR was performed using mononuclear cells (MNC) isolated from whole blood by density gradient separation. T-cell proliferation after 5-day culture was measured by (3)H-thymidine incorporation. Combined MNC from fully mismatched and partially mismatched animal pairs exhibited significant in vitro T-cell proliferation above single cell controls (P < 0.01). MNC from fully DRB-matched (but unrelated) animal pairs exhibited no proliferation compared with controls (P= 0.3). Using DRB genotyping, suitably alloreactive donor-recipient pairs may therefore be rapidly and accurately identified for use in further studies of cellular and solid organ transplantation.

The work of the Drugs Intelligence Laboratory, Home Office Forensic Science Service.

The primary purpose of the Drugs Intelligence Laboratory is to help the police identify sources of supply and manufacture of illicit drugs. This is done by providing scientific support to the Central Drugs and Illegal Immigration Intelligence Unit, a national police unit based at New Scotland Yard, London. The Laboratory provides detailed information on United Kingdom drug seizures and data obtained from a programme of comparison and characterization of seized samples. This intelligence information is then used to supplement that obtained through normal police channels in the search for illicit drug sources, manufacturers or distributors. This paper describes the techniques used for comparing and characterizing various drug preparations and outlines the type of information that can be gained from such studies.

Characterization in mice of the immunological properties of five allergenic acid anhydrides.

Occupational exposure to certain acid anhydrides, including trimellitic anhydride (TMA), maleic anhydride (MA), phthalic anhydride (PA), hexahydrophthalic anhydride (HHPA) and methyltetrahydrophthalic anhydride (MTHPA), has been associated with the development of respiratory allergy or asthma. There is considerable debate about the mechanisms through which such chemicals may cause respiratory sensitization, particularly concerning a universal requirement for specific IgE antibody. Despite the controversy regarding an obligatory role for IgE, there is a growing consensus that chemical respiratory hypersensitivity is associated with the selective development of T lymphocytes with a type 2 (Th2) phenotype. In the current investigations we have characterized in mice the nature of immune responses provoked by prolonged topical exposure to five acid anhydrides. Under application conditions where similar overall immunogenicity was achieved, we have compared cytokine responses induced by PA, MA, HHPA and MTHPA with those provoked by concurrent exposure to TMA or to the reference contact allergen 2, 4-dinitrochlorobenzene (DNCB). Lymph node cells (LNC) draining the site of topical exposure to DNCB invariably expressed high levels of the type 1 cytokines interferon-gamma (IFN-gamma) and interleukin-12 (IL-12), but only low levels of the type 2 cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10). In each experiment, TMA-activated LNC displayed the converse, type 2, phenotype of cytokine production. The other acid anhydrides in each case provoked a type 2 cytokine secretion profile, with comparable IL-10 expression but somewhat less vigorous IL-4 production compared with that observed following exposure to the reference respiratory allergen TMA. In every experiment relatively low levels of IFN-gamma and IL-12 were elaborated by acid anhydride-activated LNC, with the exception of PA-stimulated LNC that displayed increased amounts of IL-12 in comparison with other acid anhydrides. Thus, prolonged topical exposure of mice to five different acid anhydrides in each case resulted in the development of a predominantly Th2-type cytokine secretion phenotype, consistent with the ability of these materials to provoke asthma and respiratory allergy through a type 2 (possibly IgE-mediated) mechanism. Taken together with the results of previous investigations with a wider range of chemical allergens, these data suggest that induced cytokine secretion patterns or 'fingerprints' allow discrimination between contact and respiratory allergens and consequently represent a suitable approach to prospective evaluation of respiratory sensitization hazard.