Donor-Specific Antibodies Are Produced Locally in Ectopic Lymphoid Structures in Cardiac Allografts.

Cardiac allograft vasculopathy (CAV) is a transplant pathology, limiting graft survival after heart transplantation. CAV arteries are surrounded by ectopic lymphoid structures (ELS) containing B cells and plasma cells. The aim of this study was to characterize the antigenic targets of antibodies produced in ELS. Coronary arteries and surrounding epicardial tissue from 56 transplant recipients were collected during autopsy. Immunofluorescence was used to identify antibody-producing plasma cells. Immunoglobulin levels in tissue lysates were measured by enzyme-linked immunosorbent assay and analyzed for donor-specific HLA antibodies by Luminex assay. Cytokine and receptor expression levels were quantified using quantitative polymerase chain reaction. Plasma cells in ELS were polyclonal and produced IgG and/or IgM antibodies. In epicardial tissue, IgG (p < 0.05) and IgM levels were higher in transplant patients with larger ELS than smaller ELS. In 4 of 21 (19%) patients with ELS, donor-specific HLA type II antibodies were detected locally. Cytokine and receptor expression (CXCR3, interferon γ and TGF-ß) was higher in large ELS in the epicardial tissue than in other vessel wall layers, suggesting active recruitment and proliferation of T and B lymphocytes. ELS exhibited active plasma cells producing locally manufactured antibodies that, in some cases, were directed against the donor HLA, potentially mediating rejection with major consequences for the graft.

De novo alloreactive memory CD8+ T cells develop following allogeneic challenge when CNI immunosuppression is delayed.

Allospecific memory T cells are a recognized threat to the maintenance of solid-organ transplants. Limited information exists regarding the development of alloreactive memory T cells when post-transplant immunosuppression is present. The clinical practice of delaying calcineurin inhibitor (CNI) initiation post-transplant may permit the development of a de novo allospecific memory population. We investigated the development of de novo allospecific memory CD8+ T cells following the introduction of CNI immunosuppression in a murine model using allogeneic cell priming. Recipient mice alloprimed with splenocytes from fully mismatched donors received cyclosporine (CyA), initiated at 0, 2, 6, or 10days post-prime. Splenocytes from recipients were analyzed by flow cytometry or enzyme-linked immunosorbent assay for evidence of memory cell formation. Memory and effector CD8+ T cell development was prevented when CyA was initiated at 0day or 2days post-prime (p<0.001), but not 6days post-prime. Following a boost challenge, these memory CD8+ T cells were capable of producing a similarly sized population of secondary effectors as recipients not treated with CyA (p>0.05). Delaying CyA up to 6days or later post-prime permits the development of functional de novo allospecific memory CD8+ T cells. The development of this potentially detrimental T cell population in patients could be prevented by starting CNI immunosuppression early post-transplant.

Mass spectrometry measurement of a therapeutic peptide for use in multiple sclerosis.

Multiple sclerosis is an autoimmune disease of the central nervous system believed to be mediated by pathogenic T lymphocytes. We have developed a next-generation therapy in which cells secrete specific therapeutic molecules to silence these aberrant T cells. We have shown that fibroblasts, transduced to secrete a myelin basic protein-derived peptide, abrogate disease in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis, which we hypothesized using a low-zone tolerance mechanism. To determine the efficacy (or not) of this therapy in humans, we must ensure that patients receive comparable doses of therapeutic peptide. To this end, we have used liquid chromatography coupled to tandem mass spectrometry to detect a tryptic peptide, derived from the secreted therapeutic product, at nanomolar concentrations. Success depended on growing the transduced fibroblasts in defined PC-1 medium in the presence of a cocktail of protease inhibitors.

Activation of innate immunity to reduce lung metastases in breast cancer.

Breast cancer continues to be one of the leading causes of cancer death in women. Mortality is primarily due to the development of metastases. Although therapies exist, they lack efficacy in preventing metastatic growth. As a result, novel agents are being investigated. In particular, treatments that target the immune system are being examined as potential anti-neoplastic agents. Cordyceps sinensis (Cs) is a fungus that has been used for over 2,000 years in China as a treatment for a variety of conditions including neoplasms. The available evidence suggests that efficacy of Cs as an anti-neoplastic therapeutic agent is related to a role as an activator of innate immune responses. The objectives of this study were: to investigate the ability of Cs to activate macrophages to produce factors that will induce protective responses against tumour growth; to study the ability of Cs to reduce primary tumour growth in vivo; and to examine the ability of Cs to reduce lung metastasis growth in vivo. We found that oral Cs does not reduce primary tumour growth but can reduce lung metastasis occurrence in a surgical excision model of metastatic mammary carcinoma. The evidence we have shown to date suggests that the reduction in metastases growth may be due to the effects of macrophage-derived factors on tumour cell cycle.

Selection of natural health products for clinical trials: a preclinical template.

In preparation for a clinical trial on the efficacy of Echinacea products with a pediatric population, a rational method for selection of test products was developed, based on phytochemical and bioassay evaluation. Ten currently available commercial products of Echinacea angustifolia (EA) or Echinacea purpurea (EP) were selected, and 3 bottles of each of 2 different lots were purchased for each product. Investigators were blinded to product identity before phytochemical analysis. Lot-to-lot variation was small, but product variation due to species and formulation was large. Products derived from ethanol extracts had low polysaccharide content and high levels of alkamides (EA), echinacoside (EA), cynarin (EA), cichoric acid (EP), and caftaric acid (EP). These products possessed high antiviral activities that differed between EA and EP products, but limited immune activation properties. In contrast, products derived without ethanol extraction had higher polysaccharide levels, but low levels of other components. These aqueous compounds showed immunostimulant activity as measured in a mouse macrophage model and a somewhat different antiviral profile. The choice of Echinacea product for clinical trial must therefore consider the impact of immune enhancement, the specific viral infection targeted, and the potential to reduce symptoms via antiinflammatory activity. Product selection may also depend on whether the intent of the trial is prophylaxis or treatment.

The acute effects of synthetic intravenous Delta9-tetrahydrocannabinol on psychosis, mood and cognitive functioning.

BACKGROUND: Recent work suggests that heavy use of cannabis is associated with an increased risk of schizophrenia-like psychosis. However, there is a dearth of experimental studies of the effects of the constituents of cannabis, such as Delta9-tetrahydrocannabinol (THC). In a study of intravenous (i.v.) synthetic THC in healthy humans, we aimed to study the relationship of the psychotic symptoms induced by THC to the consequent anxiety and neuropsychological impairment. METHOD: Twenty-two healthy adult males aged 28+/-6 years (mean+/-s.d.) participated in experimental sessions in which i.v. THC (2.5 mg) was administered under double-blind, placebo-controlled conditions. Self-rated and investigator-rated measurements of mood and psychosis [the University of Wales Institute of Science and Technology Mood Adjective Checklist (UMACL), the Positive and Negative Syndrome Scale (PANSS) and the Community Assessment of Psychic Experiences (CAPE)] were made at baseline and at 30, 80 and 120 min post-injection. Participants also completed a series of neuropsychological tests [the Rey Auditory Verbal Learning Task (RAVLT), Digit Span, Verbal Fluency and the Baddeley Reasoning Task] within 45 min of injection. RESULTS: THC-induced positive psychotic symptoms, and participant- and investigator-rated measurements of these were highly correlated. Participants showed an increase in anxiety ratings but there was no relationship between either self- or investigator-rated positive psychotic symptoms and anxiety. THC also impaired neuropsychological performance but once again there was no relationship between THC-induced positive psychotic symptoms and deficits in working memory/executive function. CONCLUSIONS: These findings confirm that THC can induce a transient, acute psychotic reaction in psychiatrically well individuals. The extent of the psychotic reaction was not related to the degree of anxiety or cognitive impairment.

C. sinensis ablates allograft vasculopathy when used as an adjuvant therapy with cyclosporin A.

Immunosuppressive treatments are available to suppress acute cardiac rejection; however, no viable treatment exists for long-term cardiac graft failure. Moreover, extended use of calcineurin inhibitor immunosuppressants, the mainstay of the current therapeutic for cardiac transplantation, leads to significant associated pathologies such as nephrotoxicity and increased risk of cardiac disease. For the last ten years alternatives to calcineurin inhibitors, or adjuvant therapies designed to complement their activities, have been explored. In tandem with this development, there has been considerable interest in Traditional Chinese Medicines (TCM) as sources for novel therapeutics. Our study examines the ability of the TCM Cordyceps sinensis to reduce acute and chronic rejection associated with cardiac transplantation. The objectives of this study were to first determine if oral delivery of the extract could reduce acute rejection in a rat heterotopic heart model of transplantation. The second objective was to determine, in vitro, if a sterile, aqueous extract of C. sinensis could decrease CD8+ T cell activity. The third objective was to determine if oral delivery of the extract could ablate allograft vasculopathy in a mouse abdominal aortic transplant model. We found that oral delivery of the extract demonstrated a reduction in acute rejection when used in conjunction with a sub-therapeutic dose of Cyclosporine. Further, we found, using a mixed lymphocyte reaction, that the extract was able to significantly reduce CD8+ T cell activity. Finally, we demonstrate that oral delivery of the extract, used with a therapeutic dose of Cyclosporine to suppress acute rejection, ablates allograft vasculopathy.

Immune activation by a sterile aqueous extract of Cordyceps sinensis: mechanism of action.

Cordyceps sinensis is a fungus that has been used for over 2,000 years in China as a treatment for a variety of conditions including infectious diseases. The available evidence suggests a hypothesis that any efficacy of C. sinensis as an anti-infective therapeutic would be related to a role as an activator of innate immune responses. The objectives of this study were first to investigate the ability of C. sinensis to activate pro-inflammatory responses in macrophages in vitro and induce protective responses against intracellular pathogens in vivo, and second to characterize a method of action. We found that C. sinensis activates murine macrophages to produce a variety of pro-inflammatory cytokines. IFN-gamma synergizes with C. sinensis to amplify this response. Bacterial endotoxin contamination was ruled out as a potential artefact. The evidence presented in this study supports a hypothesis that C. sinensis activates macrophages by engaging Toll-like receptors and inducing mitogen-activated protein kinase (MAPK) pathways characteristic of inflammatory stimuli.

Validation of high-performance liquid chromatographic-mass spectrometric method for the analysis of lidocaine in human plasma.

A sensitive and simple liquid chromatography-tandem mass spectrometry method is developed and validated for the determination of lidocaine in human plasma. Bupivacaine is used as an internal standard, and the plasma extraction is performed by a simple liquid-liquid extraction. The limit of quantitation (LOQ) is 0.5 ng/mL with a signal-to-noise ratio greater than 5. The calibration curve is linear from 0.5 to 250 ng/mL with an r2 greater than 0.99. The coefficients of variation for within- and between-assay imprecision, including LOQ, are < or = 13% and < or = 8%, respectively. The percentage of inaccuracy for within- and between-assay, including LOQ, are < or = 9% and < or = 5%, respectively. The absolute recovery of lidocaine and bupivacaine are greater than 84% and 82%, respectively. The higher sensitivity and accuracy of this method allow the measurement of low concentrations of lidocaine in plasma from a clinical study of topically applied lidocaine in healthy subjects.

HLA-DRB1 genotyping in patients with juvenile idiopathic arthritis in Taiwan.

The object of this study was to investigate whether there is an association between HLA-DRB1 alleles and the development of juvenile idiopathic arthritis (JIA) in Taiwan. HLA-DRB1 alleles were studied in 60 patients with JIA and 200 healthy controls using polymerase chain reaction (PCR)/sequence-specific oligonucleotide probes (SSO). The frequency of HLA-DRB1*0405 in patients with JIA was found to be significantly higher than that in healthy controls [odds ratio (OR) 2.64, 95% confidence interval (CI) 1.01-6.91]. The DRB1*0405 allele was significantly associated with the development of both polyarthritis (OR 4.30, 95% CI 1.34-13.80) and oligoarthritis (OR 3.27, 95% CI 1.01-10.58). The frequency of HLA-DRB1*1502 was higher in Taiwanese JIA patients with systemic arthritis than in controls (OR 18.09, 95% CI 2.25-145.73). We conclude that, in Taiwan, HLA-DRB1*0405 is associated with the development of polyarthritis and oligoarthritis in children, and HLA-DRB1*1502 is associated with the development of systemic arthritis.

Efficacy of a standardized echinacea preparation (Echinilin) for the treatment of the common cold: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Recently, echinacea has regained popularity as one of the treatments chosen most commonly by consumers with the expectation that it will reduce the severity and duration of the common cold. However, the results from a limited number of clinical trials for this application have thus far been inconclusive. This incongruity may be the result of investigators utilizing poorly standardized echinacea products, likely devoid of sufficient quantities of active constituents necessary to exert a definitive clinical effect. Therefore, a formulation containing alkamides, cichoric acid, and polysaccharides at concentrations of 0.25, 2.5, and 25 mg/mL, respectively, was prepared from freshly harvested Echinacea purpurea plants (commercially available as Echinilin, Natural Factors Nutritional Products, Inc., Vancouver, BC, Canada). The objective of this study was to test the efficacy of this highly standardized formulation in reducing the severity and duration of symptoms of a naturally acquired common cold. METHODS: In a randomized, double-blind, placebo-controlled trial, 282 subjects aged 18-65 years with a history of two or more colds in the previous year, but otherwise in good health, were recruited. The subjects were randomized to receive either echinacea or placebo. They were instructed to start the echinacea or placebo at the onset of the first symptom related to a cold, consuming 10 doses the first day and four doses per day on subsequent days for 7 days. Severity of symptoms (10-point scale: 0, minimum; 9, maximum) and dosing were recorded daily. A nurse examined the subjects on the mornings of days 3 and 8 of their cold. RESULTS: A total of 128 subjects contracted a common cold (59 echinacea, 69 placebo). The total daily symptom scores were found to be 23.1% lower in the echinacea group than in placebo in those who followed all elements of the study protocol (P<0.01). Throughout the treatment period, the response rate to treatments was greater in the echinacea group. A few adverse event profiles were observed in both groups. CONCLUSIONS: Early intervention with a standardized formulation of echinacea resulted in reduced symptom severity in subjects with naturally acquired upper respiratory tract infection. Further studies with larger patient populations appear to be warranted.

The relative proportions of sirolimus metabolites in blood using HPLC with mass-spectrometric detection.

Measurement of sirolimus in blood as a guide to dose adjustment is an accepted practice. To date, most data have resulted from the use of a chromatographic technique. With the imminent introduction of an immunoassay into this field, there is a need to know whether metabolites that could interfere with the performance of this assay, causing a bias compared with measurements made by a chromatographic assay, vary over a period of time or with changes in concomitant immunosuppressive therapy. This preliminary study measured several sirolimus metabolites in blood samples from a variety of clinical settings, using high-performance liquid chromatography with tandem mass-spectrometric detection. Two metabolites known to cross-react in one immunoassay system, single hydroxylation products and 41-O-demethyl rapamycin, were found to constitute the bulk of the metabolic products. They were also found to form a remarkably stable proportion of all metabolites measured, both with respect to the time since transplantation and the concomitant use of cyclosporine or tacrolimus. It is concluded that the analytical bias due to cross-reactivity with metabolites, inherent in this immunoassay, should be consistent across a wide spectrum of patients receiving the drug.

Therapeutic monitoring of sirolimus: its contribution to optimal prescription.

It is now common practice to measure immunosuppressive drugs in blood as a guide to therapy. The immunosuppressive drug sirolimus, recently approved for use following kidney transplantation, was developed in the context of this clinical approach. Throughout the early clinical studies, validated analytical techniques based on chromatographic techniques were used to measure the drug. After a brief period in which an immunoassay was available, routine measurements are again being performed by chromatographic assays. In this article the use of blood concentration measurements in the assessment of the early and pivotal clinical trials of the drug is documented. Then, the rationale for the routine monitoring of the drug in clinical practice, a regulatory requirement in some countries, is set out. It is concluded that the development of this compound has benefited from experience gained during the pharmacokinetic assessment of other immunosuppressive drugs. The pharmacokinetic data accumulated on sirolimus have been a key element in formulating guidelines on dosing with this drug, both when used in combination with cyclosporine and when used after cyclosporine withdrawal.

The association of HLA -A, -B, and -DRB1 genotypes with Graves' disease in Taiwanese people.

Graves' disease has been associated with different human leukocyte antigen (HLA) genes in different races. To evaluate the association of HLA type in Taiwanese with Graves' disease, the HLA-A, -B, and -DRB1 alleles in a total of 236 Taiwanese adults with Graves' disease and 533 racially matched normal control subjects were examined using the PCR-SSOP (sequence specific oligonucleotide probe) technique. The prevalence of HLA-A*0207, -B*2704, -B*4601, and -DRB1*0901 among patients with Graves' disease was found to be increased, with odds ratios (OR) of 2.21, 3.82, 1.76 and 1.62, respectively. However, after correction for multiple comparisons, the relative risk of HLA-A*0207 susceptibility to Graves' disease remained statistically significant and the haplotype HLA-A*3303 -B*5801 -DRB1*0301 had a significantly protective effect. None of the other 2- or 3-locus haplotypes showed any significantly increased risk. Although HLA-DRB1*1405 showed an increased relative risk in patients with GO (Graves' opthalmopathy) (OR 4.61) when compared with patients without GO, the relative risk after adjusting for the number of comparisons was not significant. Taiwanese patients with Graves' disease have HLA-associated susceptibility genes which are similar to those found in Chinese patients in Hong Kong and Singapore. However, the finding in this study of a higher frequency of HLA-A*0207 in Taiwanese with Graves' disease has not been documented in any other ethnic group.

Low frequency of HLA-B*2706 in Taiwanese patients with ankylosing spondylitis.

The presence of HLA-B27 in patients affected with ankylosing spondylitis (AS) was well established prior to the advent of DNA typing of various genes within the major histocompatibility complex (MHC) in humans. However, molecular typing of the MHC genes revealed that B27 comprises a motley assortment of alleles, some of which are strongly positively associated with the disease and some of which are negatively associated with the disease. B*2706 was reported to have a negative association with AS in the Thai population and in Chinese Singaporeans. We report here our finding of an absence of B*2706 in 184 Taiwanese AS patients.

Biochemical and genetic analyses of the U5, U6, and U4/U6 x U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae.

We have purified the yeast U5 and U6 pre-mRNA splicing small nuclear ribonucleoproteins (snRNPs) by affinity chromatography and analyzed the associated polypeptides by mass spectrometry. The yeast U5 snRNP is composed of the two variants of U5 snRNA, six U5-specific proteins and the 7 proteins of the canonical Sm core. The U6 snRNP is composed of the U6 snRNA, Prp24, and the 7 Sm-Like (LSM) proteins. Surprisingly, the yeast DEAD-box helicase-like protein Prp28 is stably associated with the U5 snRNP, yet is absent from the purified U4/U6 x U5 snRNP. A novel yeast U5 and four novel yeast U4/U6 x U5 snRNP polypeptides were characterized by genetic and biochemical means to demonstrate their involvement in the pre-mRNA splicing reaction. We also show that, unlike the human tri-snRNP, the yeast tri-snRNP dissociated upon addition of ATP or dATP.

Murine hypodense eosinophils induce tumour cell apoptosis by a granzyme B-dependent mechanism.

PURPOSE: Eosinophils have been shown to potentiate anti-tumour cytotoxicity in both clinical and animal studies. The mechanism by which eosinophils induce tumour cell damage, however, has largely been speculative. The purpose of this study was to identify the mechanisms involved in eosinophil-induced tumour cell cytotoxicity. METHODS: To investigate eosinophil cytotoxicity, eosinophils were isolated from the peritoneal cavity of Mesocestoides corti-infected BALB/c mice, and were separated into normodense (ND) and hypodense (HD) populations using discontinuous Percoll density gradient centrifugation. The tumoricidal activity of ND and HD eosinophils was assessed using the [51Cr]-release cytotoxicity assay (a measure of cytolytic activity) and the JAM assay (a measure of apoptotic activity). Investigation of apoptosis-inducing molecules in HD eosinophils was undertaken by RT-PCR. The calcium chelator EGTA, serine protease inhibitor aprotinin and a competitive substrate for granzyme B were used to assess the role of perforin and granzyme B in HD eosinophil killing. RESULTS: Cytotoxic activity induced by HD eosinophils was significantly greater than that of ND eosinophils, and apoptosis was the principal killing mechanism. RT-PCR analysis revealed that HD eosinophils express mRNA for perforin, granzyme B and Fas ligand. Furthermore, HD eosinophil killing was markedly inhibited by EGTA, intracellular aprotinin and the granzyme B competitive substrate. CONCLUSIONS: These data are consistent with a hypothesis that murine HD eosinophils elicit tumoricidal activity via a granzyme B-dependent mechanism.

Prevention of allograft heart valve failure in a rat model.

OBJECTIVE: Allograft heart valves are commonly used in cardiac surgery. Despite mounting evidence that these valves are immunogenic, leading to premature failure, current clinical practice does not attempt to minimize or control such a response. The objective of this study was to evaluate immune modulatory approaches to ameliorate allograft valve failure in a rat model. METHOD: Aortic valve grafts were implanted infrarenally into Lewis rat recipients (n = 32). There were 4 transplant groups: syngeneic grafts (Lewis to Lewis), untreated allografts (Brown Norway to Lewis), allograft recipients treated with cyclosporine (INN: ciclosporin) (10 mg/kg per day for 7 or 28 days), and allograft recipients treated with anti-alpha4 integrin and anti-beta2 integrin monoclonal antibodies for 7 days. At 7 and 28 days the valves were examined for structural integrity and cellular infiltration. RESULTS: Both cyclosporine and anti-alpha4/beta2 integrin treatment resulted in significant reduction in leaflet infiltration by macrophages (ED1(+)), T cells (CD3(+)), and CD8(+) T cells at 7 days with preservation of structural integrity when compared with control allografts. Twenty-eight days after implantation, daily treatment with cyclosporine preserved leaflet structural integrity and inhibited cellular infiltration. However, a short course of cyclosporine (7 days) failed to prevent destruction of the valves at 28 days. CONCLUSIONS: Immune modulatory approaches aimed at T-cell activation or trafficking decrease leaflet cellular infiltration and prevent allograft valve structural failure. However, short-course therapy does not appear to be sufficient and must be maintained to allow long-term preservation of leaflet structural integrity (28 days).

Oral exposure to alloantigen generates intragraft CD8+ regulatory cells.

We have previously reported that oral administration of allogeneic rat spleen cells before kidney allotransplantation significantly prolongs graft survival. This prolongation was alloantigen specific and was associated with a decrease in graft-infiltrating cells (GIC) and an increase in transcription of IL-4 mRNA in the GIC. In this study increased splenic mixed lymphocyte responses from animals orally exposed to alloantigen before kidney transplantation suggested that the kidney allograft prolongation was not due to a masking of allorecognition, but to an immunomodulation of the immune response. We have assessed GIC T cell subsets on day 5 post-transplant and found decreased numbers of CD4(+) T cells in fed animals compared with controls, but there was no change in CD8(+) T cell numbers. The CD8(+) GIC from fed animals transcribed substantial levels of perforin, granzyme, and Fas ligand mRNA, indicating the presence of active CTL. Direct CTL assays showed that the GIC from fed recipients exhibited higher allo-CTL activity than GIC from control unfed recipients. In addition, the CD8(+) GIC exhibited high levels of IL-4 mRNA, suggesting Tc2-type regulatory cells. Prolonged graft survival in the face of active CTL and Tc2 cells suggests the presence of a CD8(+) regulatory cell population in the allograft. To confirm this, cell transfer experiments were performed. Prolongation of graft survival was transferred from rats orally exposed to alloantigen to naive animals by transfer of CD8(+) GIC. This is the first report that oral exposure to alloantigen prolongs kidney allograft survival by the generation of intragraft CD8(+) regulatory cells.

MoWeD, a computer program to rapidly deconvolute low resolution electrospray liquid chromatography/mass spectrometry runs to determine component molecular weights.

A computer program is described that can rapidly process low-resolution electrospray liquid chromatography/mass spectrometry (LC/MS) for peptides and proteins and assign molecular weights for observed components. The program first analyzes individual scans using a deconvolution algorithm similar to that previously described by Zhang and Marshall. Results for the entire run are then sorted by mass and those values found in adjacent scans are grouped together. The list of found components can also be compared to a user defined list of target molecular weight values making it easy to compare the results from different analyses. The program also has the capability to process a rolling average of scans that improves the performance when analyzing high molecular weight components. Other program features facilitate closer examination of selected spectra or regions of the chromatogram to check the MoWeD mass assignments. The utility of the program was demonstrated by the analysis of LC/MS data derived from a complex mixture of proteins derived from a bacterial whole cell lysate that had previously been analyzed manually. The MoWeD analysis was 30 times faster and provided a more comprehensive list of the components present.