Hepatocellular carcinoma (HCC) usually recurs after curative surgical resection. Currently, no approved adjuvant therapy has been shown to reduce HCC recurrence rates. In this study, the in vivo ...effect of sequential combination treatment with recombinant mouse interferon-alpha (rmIFN-α) and an anti-mouse-PD1 antibody on hepatitis B virus (HBV) clearance in mice was evaluated. A Phase I clinical trial was then conducted to assess the safety, tolerability, and inhibitory activity of sequential therapy with ropeginterferon alfa-2b and nivolumab in patients with HCC recurrence who underwent curative surgery for HBV-related HCC. The animal modeling study showed that HBV suppression was significantly greater with the rmIFN-α and anti-PD1 sequential combination treatment in comparison with sole treatment with rmIFN-α or anti-PD1. In the Phase I study, eleven patients completed the sequential therapy with ropeginterferon alfa-2b every two weeks for six doses at 450 µg, followed by three doses of nivolumab every two weeks up to 0.75 mg/kg. A notable decrease in or clearance of HBV surface antigen was observed in two patients. The dose-limiting toxicity of grade 3 alanine transaminase and aspartate aminotransferase increases was observed in one patient. The maximum tolerated dose was then determined. To date, no HCC recurrence has been observed. The treatment modality was well tolerated. These data support the further clinical development of sequential combination therapy as a post-surgery prophylactic measure against the recurrence of HBV-related HCC.
For sensors detecting immobilized biomarkers, the interface between the surface and the fluid medium plays an important role in determining the levels of signal and noise in the electrochemical ...detection process. When protein is directly immobilized on the metal electrode, denaturation of the protein by surface–protein interaction results in low activity and low signal level. A conducting polymer‐based interface can prevent the protein conformation change and alleviate this problem. A DNA dendrimer is introduced into the interfacial film on the sensor surface to further improve the sensor performance. DNA dendrimer is a nanoscale dendrite constructed of short DNA sequences, which can be easily incorporated into the abiotic conducting polymer matrix and is biocompatible with most biological species. In this work, DNA dendrimer and polypyrrole (DDPpy) form the bio/abiotic interface on electrochemical sensors. Detection of two salivary protein markers (IL‐8 and IL‐1β) and one mRNA salivary marker (IL‐8) is used to demonstrate the efficiency of the DDPpy sensor. A limit of detection (LOD) of protein of 100–200 fg mL−1 is achieved, which is three orders of magnitude better than that without the DNA dendrimer interface. An LOD of 10 aM is established for IL‐8 mRNA. The typical sample volume used in the detection is 4 µL, thus the LOD reaches only 25 target molecules (40 yoctomole).
A DNA dendrimer is introduced into the interfacial film on the surface of an electrochemical sensor (see picture) to improve its performance in detecting immobilized biomarkers. Limits of detection of 100–200 fg mL−1 for interleukin‐8 (IL‐8)/IL‐1β protein and of 10 am for IL‐8 mRNA are achieved. These salivary markers can detect oral cancer.
Shorter, more effective treatments for tuberculosis (TB) are urgently needed. While many TB drugs are available, identification of the best regimens is challenging because of the large number of ...possible drug-dose combinations. We have found consistently that responses of cells or whole animals to drug-dose stimulations fit a parabolic response surface (PRS), allowing us to identify and optimize the best drug combinations by testing only a small fraction of the total search space. Previously, we used PRS methodology to identify three regimens (PRS Regimens I-III) that in murine models are much more effective than the standard regimen used to treat TB. However, PRS Regimens I and II are unsuitable for treating drug-resistant TB and PRS Regimen III includes an experimental drug. Here, we use PRS methodology to identify from an expanded pool of drugs new highly effective near-universal drug regimens comprising only approved drugs.
We evaluated combinations of 15 different drugs in a human macrophage TB model and identified the most promising 4-drug combinations. We then tested 14 of these combinations in Mycobacterium tuberculosis-infected BALB/c mice and chose for PRS dose optimization and further study the two most potent regimens, designated PRS Regimens IV and V, consisting of clofazimine (CFZ), bedaquiline (BDQ), pyrazinamide (PZA), and either amoxicillin/clavulanate (AC) or delamanid (DLM), respectively. We then evaluated the efficacy in mice of optimized PRS Regimens IV and V, as well as a 3-drug regimen, PRS Regimen VI (CFZ, BDQ, and PZA), and compared their efficacy to PRS Regimen III (CFZ, BDQ, PZA, and SQ109), previously shown to reduce the time to achieve relapse-free cure in mice by 80% compared with the Standard Regimen (isoniazid, rifampicin, PZA, and ethambutol). Efficacy measurements included early bactericidal activity, time to lung sterilization, and time to relapse-free cure. PRS Regimens III-VI all rapidly sterilized the lungs and achieved relapse-free cure in 3 weeks (PRS Regimens III, V, and VI) or 5 weeks (PRS Regimen IV). In contrast, mice treated with the Standard Regimen still had high numbers of bacteria in their lungs after 6-weeks treatment and none achieved relapse-free cure in this time-period.
We have identified three new regimens that rapidly sterilize the lungs of mice and dramatically shorten the time required to achieve relapse-free cure. All mouse drug doses in these regimens extrapolate to doses that are readily achievable in humans. Because PRS Regimens IV and V contain only one first line drug (PZA) and exclude fluoroquinolones and aminoglycosides, they should be effective against most TB cases that are multidrug resistant (MDR-TB) and many that are extensively drug-resistant (XDR-TB). Hence, these regimens have potential to shorten dramatically the time required for treatment of both drug-sensitive and drug-resistant TB. If clinical trials confirm that these regimens dramatically shorten the time required to achieve relapse-free cure in humans, then this radically shortened treatment has the potential to improve treatment compliance, decrease the emergence of drug resistance, and decrease the healthcare burden of treating both drug-sensitive and drug-resistant TB.
Volatile organic compounds (VOCs) present in exhaled breath can help in analysing biochemical processes in the human body. Liver diseases can be traced using VOCs as biomarkers for physiological and ...pathophysiological conditions. In this work, we propose non-invasive and quick breath monitoring approach for early detection and progress monitoring of liver diseases using Isoprene, Limonene, and Dimethyl sulphide (DMS) as potential biomarkers. A pilot study is performed to design a dataset that includes the biomarkers concentration analysed from the breath sample before and after study subjects performed an exercise. A machine learning approach is applied for the prediction of scores for liver function diagnosis. Four regression methods are performed to predict the clinical scores using breath biomarkers data as features set by the machine learning techniques. A significant difference was observed for isoprene concentration (p < 0.01) and for DMS concentration (p < 0.0001) between liver patients and healthy subject's breath sample. The R-square value between actual clinical score and predicted clinical score is found to be 0.78, 0.82, and 0.85 for CTP score, APRI score, and MELD score, respectively. Our results have shown a promising result with significant different breath profiles between liver patients and healthy volunteers. The use of machine learning for the prediction of scores is found very promising for use of breath biomarkers for liver function diagnosis.
We report in this work successful isolation and cryopreservation of enterocytes from human small intestine. The enterocytes were isolated by enzyme digestion of the intestinal lumen, followed by ...partial purification via differential centrifugation. The enterocytes were cryopreserved directly after isolation without culturing to maximize retention of in vivo drug-metabolizing enzyme activities. Post-thaw viability of the cryopreserved enterocytes was consistently over 80% based on trypan blue exclusion. Cryopreserved enterocytes pooled from eight donors (four male and four female) were evaluated for their metabolism of 14 pathway-selective substrates: CYP1A2 (phenacetin hydroxylation), CYP2A6 (coumarin 7-hydroxylation), CYP2B6 (bupropion hydroxylation), CYP2C8 (paclitaxel 6
-hydroxylation), CYP2C9 (diclofenac 4-hydroxylation), CYP2C19 (
-mephenytoin 4-hydroxylation), CYP2D6 (dextromethorphan hydroxylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4 (midazolam 1'-hydroxylation and testosterone 6
-hydroxylation), CYP2J2 (astemizole O-demethylation), UDP-glucuronosyltransferase (UGT; 7-hydroxycoumarin glucuronidation), sulfotransferase (SULT; 7-hydroxycoumarin sulfation), and carboxylesterase 2 (CES2; irinotecan hydrolysis) activities. Quantifiable activities were observed for CYP2C8, CYP2C9, CYP2C19, CYP2E1, CYP3A4, CYPJ2, CES2, UGT, and SULT, but not for CYP1A2, CYP2A6, CYP2B6, and CYP2D6. Enterocytes from all 24 donors were then individually evaluated for the quantifiable drug metabolism pathways. All demonstrated quantifiable activities with the expected individual variations. Our results suggest that cryopreserved human enterocytes represent a physiologically relevant and convenient in vitro experimental system for the evaluation of intestinal metabolism, akin to cryopreserved human hepatocytes for hepatic metabolism.
Key points
The development of the lung is a highly stereotypical process, including the structured deployment of three distinct modes of branching: first side branching and then tip splitting with ...and without 90° rotation of the branching plane.
These modes are supposedly under genetic control, but it is not clear how genes could act to produce these spatial patterns.
Here, we show that cascades of branching events emerge naturally; the branching cascade can be explained by a relatively simple mathematical model, whose equations model the reaction and diffusion of chemical morphogens.
Our low‐dimensional model gives a qualitative understanding of how generic physiological mechanisms can produce branching phenomena, and how the system can switch from one branching pattern to another using low‐dimensional ‘control knobs’.
The model makes a number of experimental predictions, and explains several phenomena that have been observed but whose mechanisms were unknown.
Recent experimental work has described an elegant pattern of branching in the development of the lung. Multiple forms of branching have been identified, including side branching and tip bifurcation. A particularly interesting feature is the phenomenon of ‘orthogonal rotation of the branching plane’. The lung must fill 3D space with the essentially 2D phenomenon of branching. It accomplishes this by rotating the branching plane by 90° with each generation. The mechanisms underlying this rotation are not understood. In general, the programmes that underlie branching have been hypothetically attributed to genetic ‘subroutines’ under the control of a ‘global master routine’ to invoke particular subroutines at the proper time and location, but the mechanisms of these routines are not known. Here, we demonstrate that fundamental mechanisms, the reaction and diffusion of biochemical morphogens, can create these patterns. We used a partial differential equation model that postulates three morphogens, which we identify with specific molecules in lung development. We found that cascades of branching events, including side branching, tip splitting and orthogonal rotation of the branching plane, all emerge immediately from the model, without further assumptions. In addition, we found that one branching mode can be easily switched to another, by increasing or decreasing the values of key parameters. This shows how a ‘global master routine’ could work by the alteration of a single parameter. Being able to simulate cascades of branching events is necessary to understand the critical features of branching, such as orthogonal rotation of the branching plane between successive generations, and branching mode switch during lung development. Thus, our model provides a paradigm for how genes could possibly act to produce these spatial structures. Our low‐dimensional model gives a qualitative understanding of how generic physiological mechanisms can produce branching phenomena, and how the system can switch from one branching pattern to another using low‐dimensional ‘control knobs’. The model provides a number of testable predictions, some of which have already been observed (though not explained) in experimental work.
We have succeeded in developing hollow branching structure in vitro commonly observed in lung airway using primary lung airway epithelial cells. Cell concentration gradient is the key factor that ...determines production of the branching cellular structures, as optimization of this component removes the need for heterotypic culture. The higher cell concentration leads to the more production of morphogens and increases the growth rate of cells. However, homogeneous high cell concentration does not make a branching structure. Branching requires sufficient space in which cells can grow from a high concentration toward a low concentration. Simulation performed using a reaction-diffusion model revealed that long-range inhibition prevents cells from branching when they are homogeneously spread in culture environments, while short-range activation from neighboring cells leads to positive feedback. Thus, a high cell concentration gradient is required to make branching structures. Spatial distributions of morphogens, such as BMP-4, play important roles in the pattern formation. This simple yet robust system provides an optimal platform for the further study and understanding of branching mechanisms in the lung airway, and will facilitate chemical and genetic studies of lung morphogenesis programs.
A mixture of drugs is often more effective than using a single effector. However, it is extremely challenging to identify potent drug combinations by trial and error because of the large number of ...possible combinations and the inherent complexity of the underlying biological network. With a closed-loop optimization modality, we experimentally demonstrate effective searching for potent drug combinations for controlling cellular functions through a large parametric space. Only tens of iterations out of one hundred thousand possible trials were needed to determine a potent combination of drugs for inhibiting vesicular stomatitis virus infection of NIH 3T3 fibroblasts. In addition, the drug combination reduced the required dosage by almost equal to10-fold compared with individual drugs. In another example, a potent mixture was identified in thirty iterations out of a possible million combinations of six cytokines that regulate the activity of nuclear factor kappa B in 293T cells. The closed-loop optimization approach possesses the potential of being an effective approach for manipulating a wide class of biological systems.
We report here a novel in vitro enteric experimental system, cryopreserved human intestinal mucosa (CHIM), for the evaluation of enteric drug metabolism, drug-drug interaction, drug toxicity, and ...pharmacology. CHIM was isolated from the small intestines of four human donors. The small intestines were first dissected into the duodenum, jejunum, and ileum, followed by collagenase digestion of the intestinal lumen. The isolated mucosa was gently homogenized to yield multiple cellular fragments, which were then cryopreserved in a programmable liquid cell freezer and stored in liquid nitrogen. After thawing and recovery, CHIM retained robust cytochrome P450 (P450) and non-P450 drug-metabolizing enzyme activities and demonstrated dose-dependent induction of transcription of CYP24A1 (approximately 300-fold) and CYP3A4 (approximately 3-fold) by vitamin D
as well as induction of CYP3A4 (approximately 3-fold) by rifampin after 24 hours of treatment. Dose-dependent decreases in cell viability quantified by cellular ATP content were observed for naproxen and acetaminophen, with higher enterotoxicity observed for naproxen, consistent with that observed in humans in vivo. These results suggest that CHIM may be a useful in vitro experimental model for the evaluation of enteric drug properties, including drug metabolism, drug-drug interactions, and drug toxicity.
Overexpression of metastatic tumor antigen 1 (MTA1) was correlated with poor prognosis of hepatitis B virus (HBV)‐associated hepatocellular carcinoma (HBV‐HCC). The aim of this study was to examine ...the clinical significance of the expression of MTA1 and its exon 4‐excluded form (MTA1dE4), the most abundant spliced variant of MTA1, in patients receiving curative resection for HBV‐HCC. We collected 102 patients with HBV‐HCC and received curative resection retrospectively and examined the expressions level of total MTA1/MTA1dE4 in their paired nontumor and tumor liver tissues by using RT‐qPCR. The association between MTA1/MTA1dE4 expression and various tumor features as well as tumor recurrence was analyzed. During the median follow‐up period of 4 years, 25 patients (24.5%) showed early recurrence (within 12 months postresection) and 42 (54.5%) showed late recurrence. In Kaplan‐Meier analysis, MTA1dE4 overexpression in tumor, but not MTA1, was associated with early recurrence (P = 0.0365), but not late recurrence. In multivariate analysis, only alpha‐fetoprotein (AFP) ≥200 ng/mL (P = 0.006) and large tumor size (P = 0.027) were correlated with early recurrence. In the subgroup of patients with AFP <200 ng/mL, high MTA1dE4, but not total MTA1, expression could help predict early recurrence (P = 0.0195). In vitro, wound healing and invasion assays were performed in HCC cells, and MTA1dE4 was found to exhibit a higher ability in promoting migration and invasion of hepatoma cells than full‐length MTA1. Conclusion: MTA1dE4 expression is correlated with more aggressive tumor characteristics and might serve as a more sensitive marker for early recurrence of HBV‐HCC, especially for low‐AFP patients.
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