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In-vivo immunofluorescent imaging in cases of posterior uveitis
Medical Hypotheses 90 (2016) 48–50
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
In-vivo immunofluorescent imaging in cases of posterior uveitis Rohan Chawla ⇑, Pradeep Venkatesh Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi 110029, India
a r t i c l e
i n f o
Article history: Received 2 December 2015 Accepted 5 March 2016
a b s t r a c t In-vitro immunofluorescent assays/imaging are routinely used methods of detecting antigens. The ability to perform ocular angiography to study the choroidal and retinal vasculature in real time provides us with a unique opportunity to perform real time in-vivo immunofluorescent imaging. This unique combination of in-vivo immunofluorescent imaging and live imaging of choroidal and retinal circulation can help detect antigens of infective organisms in-vivo to diagnose causative infective aetiology in cases of choroiditis/retinitis. The following paper describes the basic designing of such an imaging platform. Ó 2016 Elsevier Ltd. All rights reserved.
Introduction
Proposed design
In-vivo fluorescent imaging has been tried in animals to detect macrophages [1], apoptosis [2], osteoblastic activity [3] and tumour antigens [4]. The basic approach in these imaging techniques is designing an active targeting probe [5]. An active targeting probe is a ligand which binds to a specific molecular target and has a fluorophore conjugated to it. The conjugation of the fluorophore should not significantly alter the binding capacity of the ligand. A major stumbling block in successful application of these techniques has been that opaque animal tissue hampers imaging. We feel that the ability to image the fundus and also perform fundus fluorescein angiography provides us with a very unique possibility of successfully designing and using in-vivo immunofluorescent imaging. There could be many possible molecular targets in the eye for application of this technology. In our opinion at present the most clinically useful targets would be antigens of possible infective agents which are implicated in causing various posterior uveitis. It is very difficult to arrive at an aetiological diagnosis in many such cases of likely infective posterior uveitis due to inability of ophthalmologists to provide sufficient tissue for microbiological/ pathological analysis from the eye. The antibody titres from the serum are also seldom helpful in arriving at a correct diagnosis. However, a test which enables us to directly detect the antigen right there in the eye (in-vivo) would open immense possibilities in managing posterior uveitis. Our proposed design is further described in detail below:
The imaging concept is based on in-vivo application of direct immunofluorescent technique. In cases of infective posterior uveitis the organism lies in the choroid or retina. Thus the target molecules would be an antigen of the suspected infective organism harboured in the patches of infective chorioretinitis. An appropriate recombinant monoclonal antibody (ligand) with its Fab fragment complimentary to the antigen of the infective organism will be used. The design of the Fab portion can be taken from available Fab fragments used for in-vitro immunofluorescent assays, for e.g. Fab used for Toxoplasma ELISA etc. The Fc portion of this modified antibody can be taken from antibodies such as Bevacizumab which are already approved for human use and are non-toxic. These two fragments can then be joined to make a complete antibody. This recombinant monoclonal antibody will need to be tagged with fluorescein or Indocyanine green (ICG) depending on primary area of interest (retinal vasculature or choroidal vasculature respectively). Indocyanine green appears more promising as it has already been used for in-vivo assays [6] and we already have ICG cameras for ICG angiography. Though the ability of these cameras to detect low intensity fluorescence may need to be enhanced. Other fluorophores which are non-toxic to humans and which can be combined with the ligand without altering its binding capacity can also be explored. We propose a combination of Fab and Fc fragments, as we are assuming that the fluorophore tagging will be done to the Fc portion of the antibody. However if fluorophore binding is possible to the Fab fragment itself without causing any loss to its antigen binding capacity then we could only use the tagged Fab fragment instead of the whole antibody. Basically what we desire is only a fluorophore tagged ligand to bind with the target. We may also use only a fluorophore tagged Fv component of the antibody [4].
⇑ Corresponding author at: Room No. 488, 4th Floor, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi 110029, India. Tel.: +91 1126593188, mobile: +91 9891052939; fax: +91 1126588919. E-mail address: [email protected] (R. Chawla). http://dx.doi.org/10.1016/j.mehy.2016.03.001 0306-9877/Ó 2016 Elsevier Ltd. All rights reserved.
R. Chawla, P. Venkatesh / Medical Hypotheses 90 (2016) 48–50
49
Fig. 1. Diagrammatic representation of the concept of in-vivo immunofluorescent imaging in cases of posterior uveitis.
This tagged antibody will be injected intravenously. Thus the possible/potential toxic effects of such a molecule would have to be looked into. Today we are using innumerable antibodies for a variety of diseases such as colon cancer [7], lung cancer etc [8]. Hence, we think designing a fluorophore tagged non-toxic antibody/antibody fragment should not be very difficult. This recombinant antibody will reach the patches of chorioretinitis through the blood circulation. The Fab portion of this antibody would strongly bind with the existing corresponding antigen. As the choroid and retina are both highly vascular and the blood retinal barrier is also breached in infective uveitis, the intravenously injected antibody should be able to reach its target. Some time will then be given for washout of the excessive non bound dye from the ocular circulation. As is true with in-vitro immunofluorescent assays, it is presumed that even after wash out of majority of the tagged antibody, the portion which would have bonded with the antigens would remain in the eye, providing us an opportunity to analyse the localisation and amount of its fluorescence. A fluorescein/indocyanine angiography camera would be used to image the remaining tagged antibody in the choroid/retina. If any other alternate fluorophore is used we may need to change the filters of the camera according to the dye. The amount of fluorescence detected may be less due to washout. Thus the imaging capabilities of the camera may need to be enhanced to detect and localise even low amounts of fluorescence. A photodetector attachment would help to quantify the amount of fluorescence and define levels of fluorescence for a positive result. The location of fluorescence would give us an idea of the location of the antigen. The amount of fluorescence would be
important – (a) for positivity and (b) for assessing the severity of the infection. While evaluating the fluorescence even after wash out, few considerations like minimal persistence of fluorescence around optic disc and previous chorioretinal scars would have to be analysed. These could be potential causes of false positivity and would need to be distinguished by correlation of area of fluorescence with the fundus image and/or intensity of fluorescence. The above concept is diagrammatically explained in Fig. 1. In the present model we may be able to confirm or reject the presence of only one micro-organism by each angiography using one tagged antibody. A good clinical history and examination may help the clinician to narrow down the list of possible causative agents to 2–3 micro-organisms or a possible class of microorganisms like fungal or viral etc. Thus it might take 2–3 angiographies to zero down on the causative agent. Still performing a few angiographies (which are minimally invasive) to help confirm or reject the presence of a particular infective agent might be better than empirically starting a cocktail of medications targeting multiple micro-organisms. For confirming or negating the presence of a class of micro-organisms, like fungi, an antigen common to many fungi may be used. As a research tool this technology may greatly help prove or disprove the presumed role of a particular micro-organism in certain patterns of uveitis like serpiginous uveitis, presumed tubercular vasculitis, presumed herpetic necrotising retinitis etc. Also once a basic platform using this idea is developed, methods can be thought of for enabling multiple organism detection in one angiography. For example, using a cocktail of antibodies against 2–3 organisms, each with a different fluorescent tag.
50
R. Chawla, P. Venkatesh / Medical Hypotheses 90 (2016) 48–50
Conclusion It is our opinion that with proper application of currently existing technology such a platform for in-vivo immunofluorescent ocular imaging in cases of posterior uveitis can be developed. Such an assay has the potential to completely change the way we diagnose infective posterior uveitis. Conflict of interest None of the authors has conflict of interest with the submission. No financial support was received for this submission. References [1] Che WT, Mahmood U, Weissleder R, Tung CH. Arthritis imaging using a nearinfrared fluorescence folate-targeted probe. Arthritis Res Ther 2005;7:R310–7.
[2] Petrovsky A, Schellenberger E, Josephson L, Weissleder R, Bogdanov A. Nearinfrared fluorescent imaging of tumor apoptosis. Cancer Res 2003;8: 1936–42. [3] Zaheer A, Lenkinski RE, Mahmood A, Jones AG, Cantley LC, Frangioni JV. In vivo near-infrared fluorescence imaging of osteobalstic activity. Nat Biotechnol 2001;19:1148–54. [4] Ramjiawan B, Maiti P, Aftanas A, et al. Noninvasive localization of tumors by immunofluorescence imaging using a single chain Fv fragment of a human monoclonal antibody with broad cancer specificity. Cancer 2000;89: 1134–44. [5] Rao J, Dragulescu-Andrasi A, Yao H. Fluorescence imaging in vivo: recent advances. Curr Opin Biotechnol 2007 Feb;18(1):17–25. Epub 2007 Jan 17. [6] Kim S, Lim YT, Soltesz EG, et al. Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat Biotechnol 2004;22:93–7. [7] Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004;350:2335–42. [8] Herbst RS, Nemunaitis JJ, Jablons DM, et al. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic nonsmall-cell lung cancer. J Clin Oncol 2004 Jun 1;22(11):2184–91.
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
In-vivo immunofluorescent imaging in cases of posterior uveitis Rohan Chawla ⇑, Pradeep Venkatesh Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi 110029, India
a r t i c l e
i n f o
Article history: Received 2 December 2015 Accepted 5 March 2016
a b s t r a c t In-vitro immunofluorescent assays/imaging are routinely used methods of detecting antigens. The ability to perform ocular angiography to study the choroidal and retinal vasculature in real time provides us with a unique opportunity to perform real time in-vivo immunofluorescent imaging. This unique combination of in-vivo immunofluorescent imaging and live imaging of choroidal and retinal circulation can help detect antigens of infective organisms in-vivo to diagnose causative infective aetiology in cases of choroiditis/retinitis. The following paper describes the basic designing of such an imaging platform. Ó 2016 Elsevier Ltd. All rights reserved.
Introduction
Proposed design
In-vivo fluorescent imaging has been tried in animals to detect macrophages [1], apoptosis [2], osteoblastic activity [3] and tumour antigens [4]. The basic approach in these imaging techniques is designing an active targeting probe [5]. An active targeting probe is a ligand which binds to a specific molecular target and has a fluorophore conjugated to it. The conjugation of the fluorophore should not significantly alter the binding capacity of the ligand. A major stumbling block in successful application of these techniques has been that opaque animal tissue hampers imaging. We feel that the ability to image the fundus and also perform fundus fluorescein angiography provides us with a very unique possibility of successfully designing and using in-vivo immunofluorescent imaging. There could be many possible molecular targets in the eye for application of this technology. In our opinion at present the most clinically useful targets would be antigens of possible infective agents which are implicated in causing various posterior uveitis. It is very difficult to arrive at an aetiological diagnosis in many such cases of likely infective posterior uveitis due to inability of ophthalmologists to provide sufficient tissue for microbiological/ pathological analysis from the eye. The antibody titres from the serum are also seldom helpful in arriving at a correct diagnosis. However, a test which enables us to directly detect the antigen right there in the eye (in-vivo) would open immense possibilities in managing posterior uveitis. Our proposed design is further described in detail below:
The imaging concept is based on in-vivo application of direct immunofluorescent technique. In cases of infective posterior uveitis the organism lies in the choroid or retina. Thus the target molecules would be an antigen of the suspected infective organism harboured in the patches of infective chorioretinitis. An appropriate recombinant monoclonal antibody (ligand) with its Fab fragment complimentary to the antigen of the infective organism will be used. The design of the Fab portion can be taken from available Fab fragments used for in-vitro immunofluorescent assays, for e.g. Fab used for Toxoplasma ELISA etc. The Fc portion of this modified antibody can be taken from antibodies such as Bevacizumab which are already approved for human use and are non-toxic. These two fragments can then be joined to make a complete antibody. This recombinant monoclonal antibody will need to be tagged with fluorescein or Indocyanine green (ICG) depending on primary area of interest (retinal vasculature or choroidal vasculature respectively). Indocyanine green appears more promising as it has already been used for in-vivo assays [6] and we already have ICG cameras for ICG angiography. Though the ability of these cameras to detect low intensity fluorescence may need to be enhanced. Other fluorophores which are non-toxic to humans and which can be combined with the ligand without altering its binding capacity can also be explored. We propose a combination of Fab and Fc fragments, as we are assuming that the fluorophore tagging will be done to the Fc portion of the antibody. However if fluorophore binding is possible to the Fab fragment itself without causing any loss to its antigen binding capacity then we could only use the tagged Fab fragment instead of the whole antibody. Basically what we desire is only a fluorophore tagged ligand to bind with the target. We may also use only a fluorophore tagged Fv component of the antibody [4].
⇑ Corresponding author at: Room No. 488, 4th Floor, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi 110029, India. Tel.: +91 1126593188, mobile: +91 9891052939; fax: +91 1126588919. E-mail address: [email protected] (R. Chawla). http://dx.doi.org/10.1016/j.mehy.2016.03.001 0306-9877/Ó 2016 Elsevier Ltd. All rights reserved.
R. Chawla, P. Venkatesh / Medical Hypotheses 90 (2016) 48–50
49
Fig. 1. Diagrammatic representation of the concept of in-vivo immunofluorescent imaging in cases of posterior uveitis.
This tagged antibody will be injected intravenously. Thus the possible/potential toxic effects of such a molecule would have to be looked into. Today we are using innumerable antibodies for a variety of diseases such as colon cancer [7], lung cancer etc [8]. Hence, we think designing a fluorophore tagged non-toxic antibody/antibody fragment should not be very difficult. This recombinant antibody will reach the patches of chorioretinitis through the blood circulation. The Fab portion of this antibody would strongly bind with the existing corresponding antigen. As the choroid and retina are both highly vascular and the blood retinal barrier is also breached in infective uveitis, the intravenously injected antibody should be able to reach its target. Some time will then be given for washout of the excessive non bound dye from the ocular circulation. As is true with in-vitro immunofluorescent assays, it is presumed that even after wash out of majority of the tagged antibody, the portion which would have bonded with the antigens would remain in the eye, providing us an opportunity to analyse the localisation and amount of its fluorescence. A fluorescein/indocyanine angiography camera would be used to image the remaining tagged antibody in the choroid/retina. If any other alternate fluorophore is used we may need to change the filters of the camera according to the dye. The amount of fluorescence detected may be less due to washout. Thus the imaging capabilities of the camera may need to be enhanced to detect and localise even low amounts of fluorescence. A photodetector attachment would help to quantify the amount of fluorescence and define levels of fluorescence for a positive result. The location of fluorescence would give us an idea of the location of the antigen. The amount of fluorescence would be
important – (a) for positivity and (b) for assessing the severity of the infection. While evaluating the fluorescence even after wash out, few considerations like minimal persistence of fluorescence around optic disc and previous chorioretinal scars would have to be analysed. These could be potential causes of false positivity and would need to be distinguished by correlation of area of fluorescence with the fundus image and/or intensity of fluorescence. The above concept is diagrammatically explained in Fig. 1. In the present model we may be able to confirm or reject the presence of only one micro-organism by each angiography using one tagged antibody. A good clinical history and examination may help the clinician to narrow down the list of possible causative agents to 2–3 micro-organisms or a possible class of microorganisms like fungal or viral etc. Thus it might take 2–3 angiographies to zero down on the causative agent. Still performing a few angiographies (which are minimally invasive) to help confirm or reject the presence of a particular infective agent might be better than empirically starting a cocktail of medications targeting multiple micro-organisms. For confirming or negating the presence of a class of micro-organisms, like fungi, an antigen common to many fungi may be used. As a research tool this technology may greatly help prove or disprove the presumed role of a particular micro-organism in certain patterns of uveitis like serpiginous uveitis, presumed tubercular vasculitis, presumed herpetic necrotising retinitis etc. Also once a basic platform using this idea is developed, methods can be thought of for enabling multiple organism detection in one angiography. For example, using a cocktail of antibodies against 2–3 organisms, each with a different fluorescent tag.
50
R. Chawla, P. Venkatesh / Medical Hypotheses 90 (2016) 48–50
Conclusion It is our opinion that with proper application of currently existing technology such a platform for in-vivo immunofluorescent ocular imaging in cases of posterior uveitis can be developed. Such an assay has the potential to completely change the way we diagnose infective posterior uveitis. Conflict of interest None of the authors has conflict of interest with the submission. No financial support was received for this submission. References [1] Che WT, Mahmood U, Weissleder R, Tung CH. Arthritis imaging using a nearinfrared fluorescence folate-targeted probe. Arthritis Res Ther 2005;7:R310–7.
[2] Petrovsky A, Schellenberger E, Josephson L, Weissleder R, Bogdanov A. Nearinfrared fluorescent imaging of tumor apoptosis. Cancer Res 2003;8: 1936–42. [3] Zaheer A, Lenkinski RE, Mahmood A, Jones AG, Cantley LC, Frangioni JV. In vivo near-infrared fluorescence imaging of osteobalstic activity. Nat Biotechnol 2001;19:1148–54. [4] Ramjiawan B, Maiti P, Aftanas A, et al. Noninvasive localization of tumors by immunofluorescence imaging using a single chain Fv fragment of a human monoclonal antibody with broad cancer specificity. Cancer 2000;89: 1134–44. [5] Rao J, Dragulescu-Andrasi A, Yao H. Fluorescence imaging in vivo: recent advances. Curr Opin Biotechnol 2007 Feb;18(1):17–25. Epub 2007 Jan 17. [6] Kim S, Lim YT, Soltesz EG, et al. Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat Biotechnol 2004;22:93–7. [7] Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004;350:2335–42. [8] Herbst RS, Nemunaitis JJ, Jablons DM, et al. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic nonsmall-cell lung cancer. J Clin Oncol 2004 Jun 1;22(11):2184–91.