A mesoporous gold biosensor to investigate immune checkpoint protein heterogeneity in single lung cancer cells.

Immune checkpoint proteins (ICPs) play a major role in a patient's immune response against cancer. Tumour cells usually express those proteins to communicate with immune cells as a process of escaping the anti-cancer immune response. Detecting the major functional immune checkpoint proteins present on cancer cells (such as circulating tumor cells or CTCs) and examining the heterogeneity in their expression at the single-cell level could play a crucial role in both cancer diagnosis and the monitoring of therapy. In this study, we develop a mesoporous gold biosensor to precisely assess ICP heterogeneity in individual cancer cells within a lung cancer model. The platform utilizes a nanostructured mesoporous gold surface to capture CTCs and a Surface Enhanced Raman Scattering (SERS) readout to identify and monitor the expression of key ICP proteins (PD-L1, B7H4, CD276, CD80) in lung cancer cells. The homogeneous and abundant pores in mesoporous 3D gold nanostructures enable increased antibody loading on-chip and an enhanced SERS signal, which are key to our single cell capture, and accurate analysis of ICPs in cancer cells with high sensitivity. Our lung cancer cell line model data showed that our method can detect single cells and analyse the expression of four lung cancer associated ICPs on individual cell surfaces during treatment. To show the potential of our mesoporous gold biosensor in analysing clinical samples, we tested 9 longitudinal Peripheral Blood Mononuclear Cells (PBMC) samples from lung cancer patient before and after therapy. Our mesoporous biosensor successfully captured single CTCs and found that the expression of ICPs in CTCs is highly heterogeneous in both pre-treatment and treated PBMC samples isolated from lung cancer patient blood. We suggest that our findings will help clinicians in selecting the most appropriate therapy for patients.

Nanophotonic immunoarray with electrochemically roughened surfaces for handheld detection of secreted PD-L1 to predict immuno-oncology efficacy.

The analysis of secreted protein biomarkers can be a useful non-invasive method of predicting or monitoring cancer therapeutic response. The increased level of soluble programmed cell death protein ligand 1 (sPD-L1) is a promising predictive biomarker for selecting patients who are likely to respond to immune checkpoint immunotherapy. The current established immunoassay for secreted protein analysis is enzyme-linked immunosorbent assay (ELISA). Yet, ELISA is generally still liable to limited detection sensitivity and restricted to bulky chromogenic readout equipment. Herein, we present a designed nanophotonic immunoarray sensor which achieved sPD-L1 analysis at high-throughput, enhanced detection sensitivity and portability. The key benefits of our nanophotonic immunoarray sensor are (i) high-throughput surface-enhanced Raman scattering (SERS) analysis of multiple samples on a singular platform; (ii) improved sPD-L1 detection sensitivity at 1 pg mL-1 (by two orders of magnitude as compared to ELISA) via electrochemically roughened gold sensor surfaces; (iii) fit for handheld SERS detection with miniaturized equipment footprint. We evaluated the analytical performance of the nanophotonic immunoarray sensor and successfully demonstrated quantitative sPD-L1 detection in a cohort of contrived human plasma samples.

Nanostructured mesoporous gold electrodes detect protein phosphorylation in cancer with electrochemical signal amplification.

Protein phosphorylation is a post-translational modification of kinase proteins that changes a protein's conformation to regulate crucial biological functions. However, the phosphorylation of protein is significantly altered during cancer progression which triggers abnormal cellular pathways and this phosphorylation can serve as an emergent diagnostic and prognostic biomarker for cancer. Herein, we develop a nanostructured mesoporous gold electrode (NMGE)-based biosensor that enables a highly sensitive detection of protein phosphorylation with electrochemical signal amplification. The biosensor comprises nanostructured mesoporous gold electrodes whose electro-conductive framework is superior to that of the nonporous electrodes. We characterize our developed nano/mesoporous gold electrode with various electrochemical methods in the presence of the [Fe(CN)6]3-/4- redox system. We find that the mesoporous gold electrode catalyzes both the oxidation and reduction processes of the [Fe(CN)6]3-/4- system and generates a current signal that is 3 times higher than that of the nonporous gold electrode. This superior signal transduction of our nano/mesoporous gold electrode is enabled through a pore-induced (i) high electrochemically active surface area and (ii) reduced impedance with a high signal to noise ratio. The assay utilizes direct adsorption of an immunoprecipitated purified BRAF protein towards the mesoporous gold electrode and thus avoids the cumbersome sensor surface functionalization. Our developed biosensor detects the phosphorylated BRAF protein with a 2.5-fold increase in sensitivity and an ≈10-fold increase in the limit of detection (LOD) in comparison with the nonporous gold electrodes. The assay also works on a wide dynamic range from 0.5 to 20 ng µL-1 of the protein which further shows its potential for clinical application. We envisage that this nanostructured mesoporous gold biosensor will be of high interest for clinical application.

Measurement of intestinal permeability using lactulose and mannitol with conventional five hours and shortened two hours urine collection by two different methods: HPAE-PAD and LC-MSMS.

Urinary excretion of two orally-administered non-metabolizable sugars, lactulose and mannitol, is a valuable marker for evaluating intestinal permeability. Usually this test involves a time consuming procedure of about 5 hour's urine collection, which makes the test incompatible to some extent. As the results are expressed as the ratio of lactulose and mannitol recovered in urine within certain time, it may be possible to get similar result despite the reduced urine collection time of 2 hours. Moreover, different laboratories do the test by different methods, which make the results incomparable between laboratories. Here, we are also trying to find the correlation between results from most commonly used methods: HPAE-PAD and LC-MSMS. The lactulose: mannitol (LM) test was performed in a cohort of Bangladeshi infants considered at-risk for environmental enteropathy. 208 urine specimens from 104 (52 male and 52 female) infants were collected at 2 and 5 hours after LM solution administration and were tested for lactulose and mannitol by two different methods, one HPAE-PAD platform and another LC-MSMS platform. Median age of the children was 15.0 months (range 6.9 to 25.8 months) and their mean weight-for-age z-score was -0.92. A higher percentage of lactulose and mannitol recovery was found in 5 hours urine collection than in the corresponding 2 hours by both HPAE-PAD and LC-MSMS method, but when results were expressed as lactulose to mannitol ratio (LMR) there was no significant difference between 2 and 5 hours urine collection in both HPAE-PAD (P = 0.138) and LC-MSMS (P = 0.099) method. LMR based on 2 hours urine collection correlated well with LMR based on traditional 5 hours urine collection (Spearman's correlation coefficient 0.578 and 0.604 respectively for HPAE-PAD and LC-MSMS). In future, LM test to assess intestinal permeability in children can be simplified by shortening the urine collection time from 5 hours to 2 hours.

Case-Control Study of Cryptosporidium Transmission in Bangladeshi Households.

BACKGROUND: Cryptosporidium is a leading contributor to diarrheal morbidity and mortality in under-5 children worldwide. As there is no vaccine and no effective drug therapy in young children for this infection, preventing infection is critical. We undertook a pilot case-control study to define the extent of person-to-person transmission of cryptosporidiosis within an urban and a rural community in Bangladesh. METHODS: We enrolled 48 case families with a Cryptosporidium-infected child aged 6-18 months. Controls were age- and sex-matched Cryptosporidium-negative children in 12 households. Children and household members were followed for 8 weeks with weekly illness survey and stool testing with quantitative polymerase chain reaction for Cryptosporidium. RESULTS: In the 24 urban case families, the secondary attack rate was 35.8% (19/53) vs 0% (0/11) in controls (P = .018, χ2 test). In contrast, in the 24 rural case families, the secondary attack rate was 7.8% (5/64) vs 0% (0/21) in controls (P = .19, χ2 test). Genotyping by gp60 demonstrated infection with the same subspecies in 5 families, and evidence of transmission in 2. Serologic response to Cryptosporidium infection was associated with younger age, longer duration of infection, and Cryptosporidium hominis gp60_IbA9G3R2 infection. CONCLUSIONS: In the urban site, the high rate of secondary infection and infection with the same subspecies within families suggests that person-to-person transmission is a major source of Cryptosporidium infection for young children living in this region. Molecular genotyping can be applied to determine transmission of Cryptosporidium in endemic regions. Further work is needed to understand the differences in parasite transmissibility and immunity to different genotypes.

Species of Cryptosporidia Causing Subclinical Infection Associated With Growth Faltering in Rural and Urban Bangladesh: A Birth Cohort Study.

Background: Cryptosporidiosis is a major cause of childhood diarrhea in low- and middle-income countries and has been linked to impairment of child growth. This study investigated the burden of cryptosporidiosis and its impact on child growth in both a rural and an urban site in Bangladesh. Methods: Pregnant women in the second trimester were identified at 2 sites in Bangladesh, 1 urban and 1 rural. Their offspring were enrolled at birth into the study (urban, n = 250; rural, n = 258). For 2 years, the children were actively monitored for diarrhea and anthropometric measurements were obtained every 3 months. Stool samples were collected monthly and during diarrheal episodes with Cryptosporidium infection and causative species determined by quantitative polymerase chain reaction assays. Results: Cryptosporidium infections were common at both sites and mostly subclinical. In the urban site, 161 (64%) children were infected and 65 (26%) had ≥2 infections. In the rural site, 114 (44%) were infected and 24 (9%) had multiple infections. Adjusted for potential confounders, cryptosporidiosis was associated with a significantly greater drop in the length-for-age z score (LAZ) at 24 months from LAZ at enrollment (Δ-LAZ), an effect greatest in the children with multiple episodes of cryptosporidiosis. The most common species in Mirpur was Cryptosporidium hominis, whereas Cryptosporidium meleagridis predominated in Mirzapur. Conclusions: Cryptosporidiosis is common in early childhood and associated with early growth faltering in Bangladeshi children. Predominant Cryptosporidium species differed between the 2 sites, suggesting different exposures or modes of transmission but similar consequences for child growth. Clinical Trials Registration: NCT02764918.

Giardia/Cryptosporidium QUIK CHEK Assay Is More Specific Than Quantitative Polymerase Chain Reaction for Rapid Point-of-care Diagnosis of Cryptosporidiosis in Infants in Bangladesh.

Background: Cryptosporidium is a major cause of childhood diarrhea. Current modes of cryptosporidiosis diagnosis involve procedures that are costly and require both a well-equipped laboratory and technical expertise. Therefore, a cost-effective, user-friendly, and rapid method for point-of-care detection of Cryptosporidium is desirable. Methods: A total of 832 diarrheal stool specimens collected from 200 children aged <2 years were tested by Giardia/Cryptosporidium QUIK CHEK, enzyme-linked immunosorbent assay (ELISA), and quantitative polymerase chain reaction (qPCR) to compare the performance of the individual techniques. We also tested for the presence of other diarrheal pathogens in qPCR-positive samples with a TaqMan Array Card (TAC) to assess whether Cryptosporidium was the sole causative agent for the diarrheal episodes. Results: Of 832 samples, 4.4% were found positive for Cryptosporidium by QUIK CHEK, 3.6% by ELISA, and 8.8% by qPCR. Using TAC-attributed Cryptosporidium diarrhea as the gold standard, the sensitivities of QUIK CHEK, ELISA, and qPCR were 92.3%, 71.8%, and 100%, respectively; the specificities were 97.1%, 94.3%, and 0%, respectively. Analysis of the qPCR-positive and QUIK CHEK-negative samples by TAC identified other enteropathogens as more likely than Cryptosporidium to be the causative agents of diarrhea. Conclusions: QUIK CHEK was more sensitive and specific than ELISA. While qPCR detected Cryptosporidium in more samples than QUIK CHEK, most of these were instances of qPCR detecting small quantities of Cryptosporidium DNA in a diarrheal episode caused by another enteropathogen. We concluded that QUIK CHEK was comparable in sensitivity and superior in specificity to qPCR for the diagnosis of Cryptosporidium diarrhea.

Genetic Diversity of Cryptosporidium hominis in a Bangladeshi Community as Revealed by Whole-Genome Sequencing.

We studied the genetic diversity of Cryptosporidium hominis infections in slum-dwelling infants from Dhaka over a 2-year period. Cryptosporidium hominis infections were common during the monsoon, and were genetically diverse as measured by gp60 genotyping and whole-genome resequencing. Recombination in the parasite was evidenced by the decay of linkage disequilibrium in the genome over <300 bp. Regions of the genome with high levels of polymorphism were also identified. Yet to be determined is if genomic diversity is responsible in part for the high rate of reinfection, seasonality, and varied clinical presentations of cryptosporidiosis in this population.

Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis.

The disease severity of Entamoeba histolytica infection ranges from asymptomatic to life-threatening. Recent human and animal data implicate the gut microbiome as a modifier of E. histolytica virulence. Here we have explored the association of the microbiome with susceptibility to amebiasis in infants and in the mouse model of amebic colitis. Dysbiosis occurred symptomatic E. histolytica infection in children, as evidenced by a lower Shannon diversity index of the gut microbiota. To test if dysbiosis was a cause of susceptibility, wild type C57BL/6 mice (which are innately resistant to E. histiolytica infection) were treated with antibiotics prior to cecal challenge with E. histolytica. Compared with untreated mice, antibiotic pre-treated mice had more severe colitis and delayed clearance of E. histolytica. Gut IL-25 and mucus protein Muc2, both shown to provide innate immunity in the mouse model of amebic colitis, were lower in antibiotic pre-treated mice. Moreover, dysbiotic mice had fewer cecal neutrophils and myeloperoxidase activity. Paradoxically, the neutrophil chemoattractant chemokines CXCL1 and CXCL2, as well as IL-1ß, were higher in the colon of mice with antibiotic-induced dysbiosis. Neutrophils from antibiotic pre-treated mice had diminished surface expression of the chemokine receptor CXCR2, potentially explaining their inability to migrate to the site of infection. Blockade of CXCR2 increased susceptibility of control non-antibiotic treated mice to amebiasis. In conclusion, dysbiosis increased the severity of amebic colitis due to decreased neutrophil recruitment to the gut, which was due in part to decreased surface expression on neutrophils of CXCR2.