a technique to detect extrapulmonary tuberculosis via superparamagnetic nanoprobes

A Technique to Detect Extrapulmonary Tuberculosis via Superparamagnetic Nanoprobes

To inoculate experimental animals with M. bovis BCG, first, reconstitute the lyophilized vaccine, or bacterial stock, in Sauton&#39;s medium, and dilute the reconstituted stock solution with saline. Next, load 100 microliters of the solution into one 1-milliliter syringe per animal, and inject the entire volume of bacteria, intradermally, into the left or right dorsal scapular skin of each mouse.
For in vivo magnetic resonance imaging of live nanoprobe injected animals, acquire baseline T2-weighted fast-spin echo images of each anesthetized experimental animal, before injecting two nanomolar of SPIO-Tb antibody probes, suspended in 200 microliters of saline into the tail vein of each mouse. Then, image the animals again, immediately after the injection, and every five minutes for the next 30 minutes.
At the end of the imaging session, quantitatively analyze the magnetic resonance images, using the signal intensity as a measurement of the defined regions of interest in comparable locations of an M. tuberculosis granuloma center, and the back muscle adjacent to a granulomatous area. Then, use the formula to calculate the relative signal enhancements, using the signal intensity measurement before, and, zero to three hours, after the injection of the contrast agents.

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Universidad de Cartagena UDEC

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. SPIO-MtbsAb synthesis
<ol>
	<li>Synthesize SPIO-conjugated 2,2&#39;-(ethylenedioxy)bisethylamine (EDBE) using previously reported methods.</li>
	<li>Synthesize SPIO-EDBE-succinic anhydride (SA).
	<ol>
		<li>Stir an alkaline solution (5 M NaOH; 10 mL)) of SPIO-EDBE and SA (1 g; 10 &#181;mol) at room temperature for 24 h.</li>
		<li>Dialyze the solution with 20 changes of 2 L of distilled water using molecular porous membrane tubing (12,000-14,000 MW cutoff). 6 h for each change.</li>
	</ol>
	</li>
	<li>Finally, add 100 &#956;L of SPIO-EDBE-SA (4 mg/mL of Fe) to 400 &#956;L of 4.5 mg/mL Mycobacterium tuberculosis surface antibody (MtbsAb) to synthesize SPIO-MtbsAb by using 1-hydroxybenzotriazole and (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate as catalysts and stir the solution at room temperature for 24 h.</li>
	<li>Finally, separate the solutions from the unbound antibody through gel filtration chromatography.</li>
	<li>Load the reaction mixture (5 mL) on a 2.5 cm &#215; 33 cm column and elute using a phosphate-buffered saline (PBS) buffer. Confirm Ab&#8211;nanoparticle complex (i.e., nanoprobe) using a bicinchoninic acid protein assay kit.</li>
</ol>
2. Particle morphology observation and relaxation tier measurement
<ol>
	<li>Examine average particle size, morphology, and size distribution using a transmission electron microscope at a voltage of 100 kV.
	<ol>
		<li>Drop-cast the composite dispersion onto a 200-mesh copper grid and air dry at room temperature before loading it onto the microscope.</li>
	</ol>
	</li>
	<li>Measure the relaxation time values (T1 and T2) of the nanoprobes using the NMR relaxometer at 20 MHz and 37.0 &#176;C &#177; 0.1 &#176;C.
	<ol>
		<li>Calibrate the relaxometer before each measurement.</li>
		<li>Record the r1 and r2 values from the eight data points generated through inversion-recovery and the Carr-Purcell-Meiboom-Gill pulse sequence, respectively, to determine r1 and r2 relaxivities.</li>
	</ol>
	</li>
</ol>
3. Cell imaging
<ol>
	<li>Cultivate human monocytes THP-1 in RPMI 1640 with 10% fetal bovine serum, 50 &#181;g/mL gentamycin sulfate, 100 units/mL penicillin G sodium, 100 &#181;g of streptomycin sulfate, and 0.25 &#181;g/mL fungizone in a 5% CO2 incubator at 37 &#176;C.</li>
	<li>Incubate SPIO-MtbsAb nanoprobes (2 mM) with 106 colony forming units (CFU) of Mycobacterium bovis BCG preincubated with 1 &#215; 107 activated monocytes in microcentrifuge tubes (1 mL) in a 5% CO2 incubator at 37 &#176;C for 1 h.</li>
	<li>Centrifuge tubes at 200 x g and discard the supernatant. Redissolve pellets in the medium (200 &#181;L).</li>
	<li>Scan the samples using a fast gradient echo pulse sequence (Repetition time (TR) = 500; Echo time(TE) = 20; Flip angle = 10&#176;) through 3.0-T MRI to determine the nanoprobe&#39;s specificity and sensitivity.</li>
</ol>
4. BCG (Bacillus Calmette&#8211;Gu&#233;rin) inoculation
<ol>
	<li>Reconstitute the lyophilized vaccine or bacterial stock in Sauton&#39;s medium and then dilute the stock with saline until properly dispersed as previously described.</li>
	<li>Inoculate a live attenuated strain of M. bovis BCG, obtained from ADIMMUNE (Taipei, Taiwan) (Connaught strain; ImmuCyst Aventis, Pasteur M&#233;rieux) at a volume of 0.1 mL/mouse (i.e., 107 CFU) intradermally into the left or right dorsal scapular skin of mice, as described previously. Inject saline into mice as a negative control. Monitor animals daily after BCG inoculation.</li>
	<li>Sacrifice animals 1 month after bacteria inoculation using carbon dioxide euthanasia. Harvest the tissue from the intradermal inoculation site. Fix the tissue in 10% formalin and embed it in paraffin for serial sections at 5-10 &#181;m. Stain tissue sections with the hematoxylin/eosin and Ziehl-Neelsen stains for acid-fast bacteria and with Berlin blue for ferric iron.</li>
</ol>
5. In vivo MRI
<ol>
	<li>Inject ketamine (80 mg/kg of body weight) and xylazine (12 mg/kg body weight) subcutaneously into mice for animal anesthesia.</li>
	<li>Inject SPIO-TbsAb probes (2 nmol/200 &#181;L) into the tail veins of mice. MR image mice before and immediately after probe injection and then every 5 min for 30 min to acquire T2-weighted fast spin-echo images (TR = 3000; TE = 90; field of view = 8).</li>
	<li>Quantitatively analyze all MR images using signal intensity (SI), a measurement of defined regions of interest in comparable locations of an Mtb granuloma center and the back muscle adjacent to a granulomatous area.</li>
	<li>Calculate relative signal enhancements using the SI measurement before (SIpre; control) and 0-3 h after (SIpost) injection of the contrast agents using the formula 
	[(SIpost - SIpre)/SIpre] &#215; 100 
	where SIpre is the SI of the lesion on the pre-enhanced scan and SIpost is the SI of the lesion on the post-enhanced scan.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-hydroxybenzotriazole</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>epichlorohydrin, 2,2&#39;-(ethylenedioxy)bis(ethylamine)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human monocytic THP-1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M. bovis BCG</td>
			<td>Pasteur M&#233;rieux</td>
			<td>Connaught strain; ImmuCyst Aventis</td>
			<td></td>
		</tr>
		<tr>
			<td>MRI</td>
			<td>GE medical Systems</td>
			<td>3.0-T, Signa</td>
			<td></td>
		</tr>
		<tr>
			<td>NMR relaxometer</td>
			<td>Bruker</td>
			<td>NMS-120 Minispec</td>
			<td></td>
		</tr>
		<tr>
			<td>SPECTRUM molecular porous membrane tubing, 12,000 -14,000 MW cut off</td>
			<td>Spectrum Laboratories Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TB surface antibody- Polyclonal Antibody to Mtb</td>
			<td>Acris Antibodies GmbH</td>
			<td>BP2027</td>
			<td></td>
		</tr>
		<tr>
			<td>transmission electron microscope</td>
			<td>JEOL</td>
			<td>JEM-2000 EX II</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Lee, C., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58227">Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection.</a> J. Vis. Exp. (2020).
This video demonstrates a technique for the detection of extrapulmonary tuberculosis. Superparamagnetic iron oxide (SPIO) nanoprobes, conjugated to Mycobacterium-specific antibodies, are injected into a mouse previously infected with Mycobacterium bovis BCG, labeling the granuloma and providing a better imaging contrast for its detection.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take Mycobacterium bovis&#160; BCG, an attenuated tuberculosis-causing bacteria, and intradermally inject it into a mouse.
An inflammatory response against the bacteria forms a granuloma comprising bacteria, epithelioid macrophages, conventional macrophages, and lymphocytes.
Perform magnetic resonance imaging or MRI. The magnetic field aligns randomly oriented protons in the tissue in its direction.
Apply a radio frequency pulse perpendicular to the magnetic field. The protons absorb energy from the pulse and tilt away from the field.
When the pulse stops, protons emit the absorbed energy, termed free-induction decay or FID, and realign with the field.
Measure the FID signal to obtain an image. The decay duration is tissue-specific, and areas with a longer decay appear lighter.
Take superparamagnetic nanoprobes conjugated to Mycobacterium-specific polyclonal antibodies and inject intravenously.
The antibodies bind to multiple epitopes on the bacteria, labeling the granuloma.
The nanoprobes shorten the decay of neighboring protons to make the granuloma appear darker, confirming mycobacterial infection.

22 Views. Una tecnica per rilevare la tubercolosi extrapolmonare tramite nanosonde superparamagnetiche

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An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

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Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

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A Technique to Detect Extrapulmonary Tuberculosis via Superparamagnetic Nanoprobes

Trascrizione

A Technique to Detect Extrapulmonary Tuberculosis via Superparamagnetic Nanoprobes