Outstanding Contributions of LAL Technology to Pharmaceutical and Medical Science: Review of Methods, Progress, Challenges, and Future Perspectives in Early Detection and Management of Bacterial Infections and Invasive Fungal Diseases

The blue blood of the horseshoe crab is a natural, irreplaceable, and precious resource that is highly valued by the biomedical industry. The Limulus amebocyte lysate (LAL) obtained from horseshoe crab blood cells functions as a surprisingly sophisticated sensing system that allows for the extremely sensitive detection of bacterial and fungal cell-wall components. Notably, LAL tests have markedly contributed to the quality control of pharmaceutical drugs and medical devices as successful alternatives to the rabbit pyrogen test. Furthermore, LAL-based endotoxin and (1→3)-β-D-glucan (β-glucan) assay techniques are expected to have optimal use as effective biomarkers, serving as adjuncts in the diagnosis of bacterial sepsis and fungal infections. The innovative β-glucan assay has substantially contributed to the early diagnosis and management of invasive fungal diseases; however, the clinical significance of the endotoxin assay remains unclear and is challenging to elucidate. Many obstacles need to be overcome to enhance the analytical sensitivity and clinical performance of the LAL assay in detecting circulating levels of endotoxin in human blood. Additionally, there are complex interactions between endotoxin molecules and blood components that are attributable to the unique physicochemical properties of lipopolysaccharide (LPS). In this regard, while exploring the potential of new LPS-sensing technologies, a novel platform for the ultrasensitive detection of blood endotoxin will enable a reappraisal of the LAL assay for the highly sensitive and reliable detection of endotoxemia.

Possibility of Japanese Cedar Pollen Causing False Positives in the Deep Mycosis Test

Because Japanese cedar pollen (JCP) contains beta-1,3-d-glucan (BG), there is concern that its lingering presence in the atmosphere, especially during its scattering period, may cause false positives in the factor-G-based Limulus amebocyte lysate (LAL) assay used to test for deep mycosis (i.e., G-test). Hence, we examined whether the LAL assay would react positively with substances contained in JCP by using the G-test to measure JCP particles and extracts. BG was purified from the JCP extract on a BG-specific affinity column, and the percentage extractability was measured using three different BG-specific quantitative methods. The G-test detected 0.4 pg BG in a single JCP particle and 10 fg from a single particle in the extract. The percentage extractability of JCP-derived BG was not significantly different among the three quantitative methods. As the JCP particles should technically have been removed during serum separation, they should be less likely to be a direct false-positive factor. However, given that the LAL-assay-positive substances in the JCP extract were not distinguishable by the three BG-specific quantitative methods, we conclude that they may cause the background to rise. Therefore, in Japan false positives arising from JCP contamination should be considered when testing patients for deep mycosis.

Pyrogen testing revisited on occasion of the 25th anniversary of the whole blood monocyte activation test

The whole blood pyrogen test invented 25 years ago, and its variant based on cryo-preserved blood one year later, brought momentum into the field of pyrogen testing, which, despite the broad application of the Limulus amebocyte lysate (LAL) assay, aka bacterial endotoxin test (BET), consumed several hundred thousand rabbits per year world-wide. The resulting international validation and lengthy acceptance and implementation process of what are called now monocyte activation tests (MATs) finally is impacting on animal numbers – at least in Europe – reducing them by more than 70% and counting. The author sees no reason for continuing any regulatory rabbit testing for pyrogens except the lack of acceptance of MATs in some regions of the world. The availability of MATs has opened also the discussion about the shortcomings of LAL/BET, namely its restriction to Gram-negative pyrogens, non-reflection of the potency of these in humans, interference and masking by many products, and animal welfare concerns for horseshoe crabs. The obvious advantages of MATs in all these respects should lead to a shift from LAL/BET to MATs. We are starting to see this for vac­cines and medical devices, but other areas like safety testing of blood transfusions, cell therapies and nanomaterials, and the assessment of air-borne pyrogens still need to grasp the opportunity provided by MATs. While the different MATs can jointly serve these needs, the whole blood MAT has some advantages as discussed here.

Metabolic Endotoxemia, Feeding Studies and the Use of the Limulus Amebocyte (LAL) Assay; Is It Fit for Purpose?

The Limulus amebocyte assay (LAL) is increasingly used to quantify metabolic endotoxemia (ME), particularly in feeding studies. However, the assay was not intended to assess plasma at levels typically seen in ME. We aimed to optimize and validate the LAL assay under a range of pre-treatment conditions against the well-established lipopolysaccharide binding protein assay (LBP). Fifteen healthy overweight and obese males aged 28.8 ± 9.1years provided plasma. The LAL assay employed a range of pre-treatments; 70 °C for 15 and 30 min and 80 °C for 15 and 30 min, ultrasonication (70 °C for 10 min and then 40 °C for 10 min), and dilution (1:50, 1:75, 1:100, and 1:200 parts) or diluted using 0.5% pyrosperse. Seventeen different plasma pre-treatment methods employed prior to the use of the LAL analytical technique failed to show any relationships with either LBP, or body mass index (BMI; obesity), the biological trigger for ME (p > 0.05 for all). As expected, BMI positively correlated with LBP (r = 0.523, p = 0.052. No relationships were observed between LAL with any of the sample pre-treatments and LBP or BMI. In its current form, the LAL assay is unsuitable for detecting levels of endotoxin typically seen in ME.

Endotoxin May Not Be the Major Cause of Postprandial Inflammation in Adults Who Consume a Single High-Fat or Moderately High-Fat Meal

ABSTRACT Background Metabolic endotoxemia is considered a cause for high-fat diet (HFD)-induced inflammation. However, convincing experimental evidence in humans is scant. Objective We determined whether a HFD or moderately HFD increases LPS and LPS-mediated cytokine production in the postprandial blood (PPB). Methods Ninety-eight volunteers (age: 37.3 ± 1.5 y) from the cross-sectional phenotyping study (PS) and 62 volunteers (age: 26.8 ± 1.2 y) from the intervention study (IS) consumed a breakfast containing 60% kcal fat (HF) and 36% kcal fat (moderately HF), respectively. For the IS, only the results from the placebo group are presented. Blood samples were probed for LPS-mediated cytokine production by incubating them with LPS inhibitor polymyxin B (PMB) for 24 h at 37°C besides the Limulus amebocyte lysate (LAL) assay. Repeated-measures ANOVA was used to compare the temporal changes of metabolic profiles and treatment outcomes. Results At least 87.5% of the plasma LPS measurements in 32 PS volunteers from each time point were below the LAL assay sensitivity (0.002 EU/mL). PMB suppressed IL-1β (P = 0.035) and IL-6 (P = 0.0487) production in the 3 h PPB of the PS after 24 h incubation at 37°C compared to the vehicle control, suggesting the presence of LPS. However, the amount of LPS did not increase the cytokine concentrations in the 3 h PPB above the fasting concentrations. Such suppression was not detected in the PPB of the IS. Treating whole blood with lipoprotein lipase (LPL) significantly (P < 0.05) increased FFA and cytokine (IL-1β, IL-6, TNF-α) concentrations in both studies. Conclusion LPS may not be the major cause of postprandial inflammation in healthy adults consuming a moderately HF meal (36% kcal fat, similar to the typical American diet) or a HF meal (60% kcal fat). Plasma FFAs may modulate postprandial inflammation. The prevailing concept of HFD-induced metabolic endotoxemia requires careful re-evaluation. The PS was registered at clinicaltrials.gov as NCT02367287 and the IS as NCT02472171.

Myeloperoxidase and Eosinophil Peroxidase Inhibit Endotoxin Activity and Increase Mouse Survival in a Lipopolysaccharide Lethal Dose 90% Model

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are cationic haloperoxidases with potent microbicidal and detoxifying activities. MPO selectively binds to and kills some Gram-positive bacteria (GPB) and all Gram-negative bacteria (GNB) tested. GNB contain endotoxin, i.e., lipopolysaccharide (LPS) comprising a toxic lipid A component. The possibility that MPO and EPO bind and inhibit the endotoxin of GNB was tested by mixing MPO or EPO with LPS or lipid A and measuring for inhibition of endotoxin activity using the chromogenic Limulus amebocyte lysate (LAL) assay. The endotoxin-inhibiting activities of MPO and EPO were also tested in vivo using an LPS 90% lethal dose (LD90) mouse model studied over a five-day period. Mixing MPO or EPO with a fixed quantity of LPS from Escherichia coli O55:B5 or with diphosphoryl lipid A from E. coli F583 inhibited LAL endotoxin activity in proportion to the natural log of the MPO or EPO concentration. MPO and EPO enzymatic activities were not required for inhibition, and MPO haloperoxidase action did not increase endotoxin inhibition. Both MPO and EPO increased mouse survival in the LPS LD90 model. In conclusion, MPO and EPO nonenzymatically inhibited in vitro endotoxin activity using the LAL assay, and MPO and high-dose EPO significantly increased mouse survival in a LPS LD90 model, and such survival was increased in a dose-dependent manner.

Microbial Profile and Endotoxin Levels in Primary Periodontal Lesions with Secondary Endodontic Involvement

This study was carried out to investigate the microbial profile and endotoxin levels of endodontic-periodontal lesions of periodontal origin. Periodontal and endodontic samples were taken from periodontal pockets and necrotic root canals of 10 teeth with endodontic-periodontal lesions. Evidencing of 40 different bacterial species were determined in each endodontic and periodontal sample using the checkerboard DNA-DNA hybridization method and Kinetic chromogenic LAL assay was used for quantification of endotoxins. Fisher’s exact test correlated the bacterial species with the endodontic or periodontal microbiota. The endotoxin levels (EU/mL) found in samples of the root canal and periodontal pocket were compared by the Wilcoxon test (p<0.05). Bacteria and LPS units were found in 100% of the endodontic and periodontal samples. The species E. faecium, P. acnes, G. morbillorum, C. sputigena and L. buccalis were strongly correlated with the endodontic microbiota and P. nigrescens with the periodontal microbiota. P. intermedia, P. endodontalis and V. parvula were more prevalent in both endodontic and periodontal microbiots. The endotoxin levels in the periodontal pocket (89600 EU/mL) were significantly higher than in the root canal (2310 EU/mL). It was concluded that the microbiota present in the periodontal and endodontic tissues is similar, with a higher prevalence of species of the orange complex and a higher level of endotoxin in the periodontal pockets.