This work was supported by NIH grant P20RR018766 and a grant from the ABMRF/Foundation for Alcohol Research.

<ol>
	<li>Chakraborty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+system:+a+vital+link+between+metabolic+syndrome+and+inflammation.">Lymphatic system: a vital link between metabolic syndrome and inflammation.</a> Ann. N. Y. Acad. Sci. 1207, 94-94 (2010).</li><li>Zawieja, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+biology+and+the+microcirculation:+past,+present+and+future.">Lymphatic biology and the microcirculation: past, present and future.</a> Microcirculation. 12, 141-141 (2005).</li><li>Benoit, J. N., Zawieja, D. C., Goodman, A. H., Granger, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+intact+mesenteric+lymphatic+pump+and+its+responsiveness+to+acute+edemagenic+stress.">Characterization of intact mesenteric lymphatic pump and its responsiveness to acute edemagenic stress.</a> Am. J. Physiol. 257, H2059-H2059 (1989).</li><li>Davis, M. J., Davis, A. M., Ku, C. W., Gashev, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+constriction+and+dilation+of+isolated+lymphatic+vessels.">Myogenic constriction and dilation of isolated lymphatic vessels.</a> Am. J. Physiol. Heart. Circ. Physiol. 296, H293-H293 (2009).</li><li>Dougherty, P. J., Davis, M. J., Zawieja, D. C., Muthuchamy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sensitivity+and+cooperativity+of+permeabilized+rat+mesenteric+lymphatics.">Calcium sensitivity and cooperativity of permeabilized rat mesenteric lymphatics.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R1524-R1524 (2008).</li><li>Fay, F. S., Shlevin, H. H., Granger, W. C., Taylor, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aequorin+luminescence+during+activation+of+single+isolated+smooth+muscle+cells.">Aequorin luminescence during activation of single isolated smooth muscle cells.</a> Nature. 280, 506-506 (1979).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+myosin+light+chain+phosphorylation+decreases+rat+mesenteric+lymphatic+contractile+activity.">Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity.</a> Am. J. Physiol. Heart. Circ. Physiol. 297, 726-726 (2009).</li><li>Karaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+movements,+distribution,+and+functions+in+smooth+muscle.">Calcium movements, distribution, and functions in smooth muscle.</a> Pharmacol. Rev. 49, 157-157 (1997).</li><li>Ratz, P. H., Berg, K. M., Urban, N. H., Miner, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+smooth+muscle+calcium+sensitivity:+KCl+as+a+calcium-sensitizing+stimulus.">Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus.</a> Am. J. Physiol. Cell. Physiol. 288, C769-C769 (2005).</li><li>Somlyo, A. P., Somlyo, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++sensitivity+of+smooth+muscle+and+nonmuscle+myosin+II:+modulated+by+G+proteins,+kinases,+and+myosin+phosphatase.">Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase.</a> Physiol. Rev. 83, 1325-1325 (2003).</li><li>Souza-Smith, F. M., Kurtz, K. M., Molina, P. E., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+mesenteric+collecting+lymphatic+pump+function+following+acute+alcohol+intoxication.">Adaptation of mesenteric collecting lymphatic pump function following acute alcohol intoxication.</a> Microcirculation. 17, 514-514 (2010).</li><li>Breslin, J. W., Yuan, S. Y., Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-C+alters+barrier+function+of+cultured+lymphatic+endothelial+cells+through+a+VEGFR-3-dependent+mechanism.">VEGF-C alters barrier function of cultured lymphatic endothelial cells through a VEGFR-3-dependent mechanism.</a> Lymphat. Res. Biol. 5, 105-105 (2007).</li><li>Shirasawa, Y., Benoit, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch-induced+calcium+sensitization+of+rat+lymphatic+smooth+muscle.">Stretch-induced calcium sensitization of rat lymphatic smooth muscle.</a> Am. J. Physiol. Heart. Circ. Physiol. 285, H2573-H2573 (2003).</li><li>Imtiaz, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pacemaking+through+Ca2++stores+interacting+as+coupled+oscillators+via+membrane+depolarization.">Pacemaking through Ca2+ stores interacting as coupled oscillators via membrane depolarization.</a> Biophys. J. 92, 3843-3843 (2007).</li><li>Muller, J. M., Davis, M. J., Kuo, L., Chilian, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+coronary+endothelial+cell+Ca2++concentration+during+shear+stress-+and+agonist-induced+vasodilation.">Changes in coronary endothelial cell Ca2+ concentration during shear stress- and agonist-induced vasodilation.</a> Am. J. Physiol. 276, 1706-1706 (1999).</li><li>Ferrusi, I., Zhao, J., van Helden, D., von der Weid, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclopiazonic+acid+decreases+spontaneous+transient+depolarizations+in+guinea+pig+mesenteric+lymphatic+vessels+in+endothelium-dependent+and+-independent.">Cyclopiazonic acid decreases spontaneous transient depolarizations in guinea pig mesenteric lymphatic vessels in endothelium-dependent and -independent.</a> 286, H2287-H2287 (2004).</li></ol>

No conflicts of interest declared.

A novel combination of methods was employed to study intrinsic pumping of lymphatic vessels. The ability to simultaneously measure changes in [Ca2+]i and diameter in pumping lymphatics will allow for studies of the relative contributions of Ca2+-dependent and Ca2+-sensitizing signaling pathways in the overall mechanism governing the lymphatic contractile cycle.
The lymphatic contractile cycle consists of phasic contractions superimposed over tone. Th...

<table border="1">
<tbody>
<tr>
<td>1. Ringer 5x Stock</td>
</tr>
<tr>
<tdChemical</td>
<td>Company</td>
<td>Catalog Number</td>
<td>Amount </td>
</tr>
<tr>
<td>Sodium Chloride</td>
<td>EMD</td>
<td>SX0420-3</td>
<td>35 g</td>
</tr>
<tr>
<td>Potassium Chloride</td>
<td>J.T. Baker</td>
<td>3040</td>
<td>1.75 g</td>
</tr>
<tr>
<td>Calcium Chloride</td>
<td>Sigma</td>
<td>C-3881</td>
<td>1.47 g</td>
</tr>
<tr>
<td>Magnesium Sulfate</td>
<td>Sigma</td>
<td>M-9397</td>
<td>1.44 g</td>
</tr>
<tr>
<td>Sterile Filtered Water</td>
<td>N/A</td>
<td>N/A</td>
<td>Bring to 1 L</td>
</tr>
<tr>
<td>Sterile filter into autoclaved bottles and stores at 4 &deg;C</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>2. MOPS buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Chemical</td>
<td>Company</td>
<td>Catalog Number</td>
<td>Amount </td>
</tr>
<tr>
<td>MOPS</td>
<td>Sigma</td>
<td>M3183</td>
<td>125.6 g</td>
</tr>
<tr>
<td>Sterile Filtered Water</td>
<td>N/A</td>
<td>N/A</td>
<td>Bring to 1 L</td>
</tr>
<tr>
<td>Sterile filter into autoclaved bottles and stores at 4 &deg;C</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>3. Albumin Physiological Salt Solution (APSS)</td>
</tr>
<tr>
<td>Chemical</td>
<td>Company</td>
<td>Catalog Number</td>
<td>Amount </td>
</tr>
<tr>
<td>Ringer stock (5x)</td>
<td>N/A</td>
<td>N/A</td>
<td>200 mL </td>
</tr>
<tr>
<td>Mops Buffer</td>
<td>N/A</td>
<td>N/A</td>
<td>5 mL </td>
</tr>
<tr>
<td>Sodium Phosphate</td>
<td>Sigma</td>
<td>S-9638</td>
<td>0.168 g</td>
</tr>
<tr>
<td>Sodium Pyruvate</td>
<td>Sigma</td>
<td>P5280</td>
<td>0.22 g</td>
</tr>
<tr>
<td>EDTA sodium salt</td>
<td>Sigma</td>
<td>ED2SS</td>
<td>0.0074 g</td>
</tr>
<tr>
<td>Glucose</td>
<td>Sigma</td>
<td>G7528</td>
<td>0.901 g</td>
</tr>
<tr>
<td>Albumin, Bovine</td>
<td>USB</td>
<td>10856</td>
<td>10 g</td>
</tr>
<tr>
<td>Sterile Filtered Water</td>
<td>N/A</td>
<td>N/A</td>
<td>Bring to 1 L</td>
</tr>
<tr>
<td>Adjust pH to 7.4 at 37&deg; C, then sterile filter into autoclaved bottles and store at 4 &deg;C.</td>
</tr>
</tbody>
</table>
Table 1. Specific Reagents Used. Store all at 4 &deg;C.

measurement of cytosolic ca2+ in isolated contractile lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Watch this Scientific Journal Video about Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics at JoVE.com

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

measurement-of-cytosolic-ca2-in-isolated-contractile-lymphatics

Research

JoVE Journal

Immunology and Infection

13.5K vues.  Louisiana State University Health Sciences Center. Nous introduisons une approche pour évaluer la cytosolique de Ca 2 + dans les vaisseaux lymphatiques isolés pour étudier Ca 2 + Ca-dépendante et 2 + Sensibilisant les mécanismes de la contraction musculaire lisse lymphatique.

Vidéo: Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

Measurement of &amp;#947;HV68 Infection in Mice

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently shown that UPEC have an important role in the initiation of chronic pelvic pain2, a feature of Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS)3,4. Infection of the prostate by clinically relevant UPEC can initiate and establish chronic pain through mechanisms that may involve tissue damage and the initiation of mechanisms of autoimmunity5.
A challenge to understanding the pathogenesis of UPEC in the prostate is the relative inaccessibility of the prostate gland to manipulation. We utilized a previously described intraurethral infection method6 to deliver a clinical strain of UPEC into male mice thereby establishing an ascending infection of the prostate. Here, we describe our protocols for standardizing the bacterial inoculum7 as well as the procedure for catheterizing anesthetized male mice for instillation of bacteria.
CP/CPPS is primarily characterized by the presence of tactile allodynia4. Behavior testing was based on the concept of cutaneous hyperalgesia resulting from referred visceral pain8-10. An irritable focus in visceral tissues reduces cutaneous pain thresholds allowing for an exaggerated response to normally non-painful stimuli (allodynia). Application of normal force to the skin result in abnormal responses that tend to increase with the intensity of the underlying visceral pain. We describe methodology in NOD/ShiLtJ mice that utilize von Frey fibers to quantify tactile allodynia over time in response to a single infection with UPEC bacteria.

Infection of the prostate may be a contributing factor in mediating pelvic pain in chronic prostatitis. We describe the procedure for preparation of standardized bacterial inoculum, instillation of bacteria into the urethra of male mice and methodology for measuring tactile allodynia in mice over time.

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. 
In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Measurement of Chitinase Activity in Biological Samples

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body including at baseline. The precise role of chitinase is not known, however it was believed to play important role in digestion and host defense against chitin-containing food and pathogens, respectively. Recent work, including ours, has shown an important role of chitinase and chitinase-like proteins in host immunity and allergic diseases. Importantly, chitinase activities serve as important biomarkers of disease severity in a wide-range of diseases including type 2 inflammatory diseases such as asthma and pulmonary fibrosis. Similarly, patients with genetic disorders like Gaucher disease have significantly elevated chitinase levels, which not only correlate with disease severity but also serve as a reliable biomarker for therapeutic effectiveness. The protocol outlined here describes a simple, quick, and straightforward way to measure chitinase activity in BAL or serum samples of mice and can be widely adapted to human subjects and other model organisms due to the highly conserved nature of the enzymes.

Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum.

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body ...