Production of attenuated leishmania donovani by nd:YAG laser and its medical application in mice as attenuated vaccine ( in vivo) for immunization with elimination of visceral leishmaniasis

Abstract

Production of attenuated L. donovani by Nd:YAG laser and its medical application in mice as attenuated vaccine (invivo) was planted on N199 semi solid media, its exposed to Nd:YAG laser in wavelength 1060°A, in 500 pulse (between each pulse 6 second), Calculated viable cells and percentage of killing using MTT stain by ELISA reader, it was killed highly. Approved results presence high percentage of killing of L. donovani which was exposed to Nd:YAG laser and the viable cells of L. donovani exposed to laser devoid to flagellum (flagellum responsible for movement, penetrate to liver and spleen for cause infection). Results approved the mice its infected with sign and symptoms of infection include fever, enlarge the size of liver and spleen (become very bulked), when done smear from liver and spleen for injected mice approve presence of L. donovani under light microscope on slide in amastigote, this results indicate the radiation emitted from Nd:YAG laser is efficient and effective in attenuation L. donovani its lost of flagellum, attenuated and not cause infection for liver and spleen in 500 pulse with 4 1060 Ao, therefor can used L. donovani exposed to radiation emitted from Nd :YAG laser as attenuated vaccine against infection of L. donovani, this method indicates to use L. donovani after exposure them to Nd:YAG laser as attenuated vaccine.

Description

[0001] Production of attenuated Leishmania donovani by Nd:YAG laser and its medical application in Mice as attenuated vaccine (in vivo) for immunization with elimination of visceral leishmaniasis

[0002] The basis of the idea was approved to include that there is no emitted radiation used to immunize organisms {in vivo) by making a weakened Leishmania vaccine exposed to a laser and used to eliminate visceral leishmaniasis, so the basis of the idea is to use the radiation emitted from an Nd: YAG laser to weaken visceral leishmaniasis. At a wavelength of 1060 °A in 500 pulses (6 seconds between each pulse) and immunizing organisms including mice with attenuated visceral leishmaniasis in vivo).

[0003] The aim of the study is to eliminate dangerous and fatal leishmaniasis after exposure to the Nd: YAG laser and to use attenuated visceral leishmaniasis as an attenuated vaccine for in vivo immunization and elimination of visceral leishmaniasis.

[0004] Nd: YAG is a pulsed laser with a wavelength of 1060 °A and at different energies. The rays emanating from it are easily absorbed by melanin and hemoglobin and the energy emanating from it can also be transmitted in water. These properties make it a suitable and excellent choice in treatment [1], Mechanical effect of laser rays on microorganisms by generating partial heat and thus the heat generated from the effect of photothermal heat instead of chemical heat, it leads to the denaturation of microbial cells and their proteins, which leads to their killing [2], Therefore, it can be used in treatment, especially since it is used in the field of medicine and sterilization of dental instruments because of its reduction of a large number of Living cells of microbes in aquatic suspension [3], Laser rays were named based on the wavelength or the active laser medium and the active media used is either gas, liquid, solid, semiconductor or biological materials. The solid-state laser media is either glass or crystalshaped, as for the active laser material that it carries. The most common host material is Yttrium aluminum-garnet (YAG) and among many active materials is Neodymium, thus the name Nd: YAG laser came about [4], The antibiotics used in the treatment of leishmaniasis: Stibgluconate, Pntamedine, and Amphotericin B, which are toxic, and giving these antibiotics requires staying for long periods in the hospital [5], They are expensive and their uses are limited. Also, these antibiotics are ineffective against several types of leishmania parasites [6], The effect of the antibiotics is either inactivation or inactivation of the leishmaniasis. Energy production or metabolism as in trypanothione [7],

[0005] Visceral leishmaniasis is caused by a primary obligatory intracellular parasite that extends to the genus Leishmania [8], There are more than 20 species under the genus Leishmania that infect humans through the vector, the sand fly, and the infection is transmitted to milkmaids through it. It is transmitted by the sand fly of the Lutzomyia species, which was considered a vector in the ancient world [9,10],

[0006] Leishmaniasis is caused by obligate parasites that invade phagocytic cells. It is endemic in large parts of the tropical and subtropical regions and in the Mediterranean basin. This disease results from infection with more than twenty types of parasites and is transmitted to humans through approximately 30 different species. From the sand fly

[0011] , it infects macrophage cells throughout the viscera, and the parasite infects the liver, spleen and bone marrow. Clinical symptoms include fever (high temperatures), chills, ulcers at the site of the bite, anemia (lack of red blood cells), Hepatosplenomegaly, weight loss, and the presence of multiple IgG and IgM clones indicate that failure to treat this disease causes death

[0012] ,

[0007] This parasite parasitizes humans and causes visceral leishmaniasis. The infection occurs mainly in the liver and spleen, but the parasite may infect other sites such as bone marrow and intestinal villi. The disease it causes is called “kalazar,” meaning the black disease. The parasite’s life cycle includes inside the retinal system. The epithelium of the viscera, which includes the spleen, liver, lymph nodes, and also bone marrow, and the leishmanial phase is found in all tissues and fluids of the body

[0013] , The clinical symptoms of leishmaniasis are not limited only to the presence of the type of parasite. They also depend on the genetic factors of the host, the health of the host, and the immune changes associated with resistance and sensitivity to the disease. All of these factors affect the design of the vaccine. Disclosure of tave ic

[0008] 1-3 The studied parasite

[0009] The study was conducted on (25) L. donovani that cause visceral leishmaniasis from different hospitals in Baghdad.

[0010] 2-3 Diagnosis

[0011] Visceral leishmania was diagnosed based on biochemical tests and its cultural and microscopic characteristics on different culture media.

[0012] 1-2-3 Agricultural media

[0013] Visceral leishmania was grown on semi-solid media (N199), a special media for leishmania growth.

[0014] 3-3 Nd:YAG laser used to attenuation visceral leishmania An Nd: YAG laser with a wavelength of 1060 A0was used in 500 pulses (6 seconds between one pulse).

[0015] 4-3 Count of viable cells and percentage of killing of visceral leishmania exposed to Nd:YAG laser

[0016] The number of live cells and the killing percentage were calculated for the visceral leishmania after exposure to the Nd: YAG laser, as well as for the positive control (Control).

[0017] The aim of the study is to eliminate dangerous and fatal leishmaniasis after exposure to the Nd: YAG laser and to use attenuated visceral leishmaniasis as an attenuated vaccine for in vivo immunization and elimination of visceral leishmaniasis.

[0018] Nd: YAG is a pulsed laser with a wavelength of 1060 °A and at different energies. The rays emanating from it are easily absorbed by melanin and hemoglobin and the energy emanating from it can also be transmitted in water. These properties make it a suitable and excellent choice in treatment [1], Mechanical effect of laser rays on microorganisms by generating partial heat and thus the heat generated from the effect of photothermal heat instead of chemical heat, it leads to the denaturation of microbial cells and their proteins, which leads to their killing [2] . Therefore, it can be used in treatment, especially since it is used in the field of medicine and sterilization of dental instruments because of its reduction of a large number of Living cells of microbes in aquatic suspension [3],

[0019] Laser rays were named based on the wavelength or the active laser medium and the active media used is either gas, liquid, solid, semiconductor or biological materials. The solid-state laser media is either glass or crystal- shaped, as for the active laser material that it carries. The most common host material is Yttrium aluminum-gamet (YAG) and among many active materials is Neodymium, thus the name Nd: YAG laser came about [4],

[0020] The antibiotics used in the treatment of leishmaniasis: Stibgluconate, Pntamedine, and Amphotericin B, which are toxic, and giving these antibiotics requires staying for long periods in the hospital [5], They are expensive and their uses are limited. Also, these antibiotics are ineffective against several types of leishmania parasites [6] . The effect of the antibiotics is either inactivation or inactivation of the leishmaniasis. Energy production or metabolism as in trypanothione [7], Visceral leishmaniasis is caused by a primary obligatory intracellular parasite that extends to the genus Leishmania [8], There are more than 20 species under the genus Leishmania that infect humans through the vector, the sand fly, and the infection is transmitted to milkmaids through it. It is transmitted by the sand fly of the Lutzomyia species, which was considered a vector in the ancient world [9,10], Leishmaniasis is caused by obligate parasites that invade phagocytic cells. It is endemic in large parts of the tropical and subtropical regions and in the Mediterranean basin. This disease results from infection with more than twenty types of parasites and is transmitted to humans through approximately 30 different species. From the sand fly

[0011] , it infects macrophage cells throughout the viscera, and the parasite infects the liver, spleen and bone marrow. Clinical symptoms include fever (high temperatures), chills, ulcers at the site of the bite, anemia (lack of red blood cells), Hepatosplenomegaly, weight loss, and the presence of multiple IgG and IgM clones indicate that failure to treat this disease causes death

[0012] ,

[0021] This parasite parasitizes humans and causes visceral leishmaniasis. The infection occurs mainly in the liver and spleen, but the parasite may infect other sites such as bone marrow and intestinal villi. The disease it causes is called “kalazar,” meaning the black disease. The parasite’s life cycle includes inside the retinal system. The epithelium of the viscera, which includes the spleen, liver, lymph nodes, and also bone marrow, and the leishmanial phase is found in all tissues and fluids of the body

[0013] , The clinical symptoms of leishmaniasis are not limited only to the presence of the type of parasite. They also depend on the genetic factors of the host, the health of the host, and the immune changes associated with resistance and sensitivity to the disease. All of these factors affect the design of the vaccine.

[0022] 1-2 Preparing and exposing visceral leishmaniasis cells L. donovani to the Nd:YAG laser and calculating the number of live cells and the killing percentage

[0023] 1-1-2 Cultivation of visceral leishmaniasis and determination of the percentage of killing

[0024] The method described by

[0016] was followed with some modifications to expose the visceral leishmania cells under test to the Nd: YAG: 1- Transferring pure cells from visceral leishmania to 150 ml cultured on semi-solid medium N199 for 5 days at 25°C.

[0025] 2- Centrifuge for ten minutes at 5000 rpm, take the pellet sediment and re-suspend it in 150 ml of sterile physiological salt solution to maintain cell vitality.

[0026] 3- Withdraw 1 ml of the suspension and expose it to an Nd: YAG laser at a wavelength of 1060 Ao in 500 pulses (6 seconds between each pulse) and compare with a control tube without exposure to radiation.

[0027] 4- Read the number of live colonies using the MTT dye using the ELISA reader, and calculate the number of live cells from the following equation:

[0028] AT - AB

[0029] Live cells % =

[0030] AC - AB * 100

[0031] 5- The killing rate was calculated from the following equation.

[0032] Control - Treated

[0033] Kill rate =

[0034] Control *100

[0035] Control: Visceral leishmania before exposing it to radiation emitted by the Nd: YAG laser.

[0036] Treated: Visceral leishmania after exposure to radiation emitted by the Nd: YAG laser.

[0037] 2-2 MTT experience

[0038] The best test used for live cell counting, designed with a 96-hole plate

[0017] is used to sift a group of compounds to determine whether the compounds used for the test have effects on cell proliferation or have a direct toxic effect that leads to cell death, and it is also used to measure A variety of assays MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) tetrazolium reduction assay.

[0039] The relative numbers of live cells are determined by the optical absorbance of treated and untreated samples as well as blank (culture medium only) using the following equation:

[0040] AT - AB

[0041] Number of live cells % =

[0042] AC - AB *100

[0043] AT: Absorbance of samples treated with laser.

[0044] AB: Absorbance of the culture medium only (Blank).

[0045] AC: Absorbance of samples not treated with laser (Control).

[0046] 1-2-2 Prepare MTT solution

[0047] 1- Dissolve 100 mg of MTT in 10 ml of distilled water (take 10 mg of MTT) in 1 ml of distilled water. 2- Filter the MTT solution using 0.2 mm filter paper.

[0048] 3- Store the MTT solution away from light at 4 °C for repeated use or in C. ° -02 for long term storage

[0049] 2-2-2 Viable cell counting examination using MTT dye

[0050] 1-A ninety six well plate was used and the promastigotes of the visceral leishmania parasite were added to it at a final size of 100 microns per hole.

[0051] 2- The plate was incubated in the incubator at 25 °C for 3 day.

[0052] 3- Add ten microns of MTT solution to the plate to obtain a final concentration of 0.45 mg / ml.

[0053] 4- The plate was incubated for four hours in the incubator at 25 °C.

[0054] 5- Remove excess culture medium and add 100 microns of DMSO solution.

[0055] 6- Gently move the plate and leave for 15 min.

[0056] 7- Reading the absorbance at the wavelength of 490 nm using the ELISA reader

[0018] ,

[0057] 2-3 Injection of attenuated visceral leishmania exposed to Nd:YAG laser in mice

[0058] 1 -Adding attenuated visceral leishmania to physiological salt solution.

[0059] 2- Exposing the visceral leishmania in the suspension to an Nd: YAG laser with 500 pulses at a wavelength of 1060 Ao (6 seconds between one pulse).

[0060] 3- Visceral leishmania was cultured after exposure in N199 medium and incubated at 25°C for four days.

[0061] 4- Examination under a microscope.

[0062] 5- Preparing a suspension using physiological salt solution and comparing its turbidity with the McFarland turbidity (NO.5) of attenuated visceral leishmania exposed to the Nd: YAG laser.

[0063] 6- Withdraw 0.2 ml (200 pl) of the suspension and inject it into the mice into the peritoneum (12 mice) and compare it with a control with duplicates.

[0064] 3- Results

[0065] 1-3 The studied parasite

[0066] The study was conducted on (25) L. donovani that cause visceral leishmaniasis from different hospitals in Baghdad.

[0067] 2-3 Diagnosis

[0068] Visceral leishmania was diagnosed based on biochemical tests and its cultural and microscopic characteristics on different culture media.

[0069] 1-2-3 Agricultural media

[0070] Visceral leishmania was grown on semi-solid media (N199), a special media for leishmania growth.

Claims

ClaimsClaims include protection for:1- The attenuated vaccine for attenuated Leishmaniadonovani produced by exposure to an Nd:YAG laser at 500 pulses at a wavelength of 1060 Aoused in attenuation and applied to mice as an attenuated vaccine (AT - ABNumber of live cells % =AC - AB * 100AT: Absorbance of samples treated with laser.AB: Absorbance of the culture medium only (Blank).AC: Absorbance of samples not treated with laser (Control).-Dissolve 100 mg of MTT in 10 ml of distilled water (take 10 mg ofMTT) in 1 ml of distilled water.- Filter the MTT solution using 0.2 mm filter paper.- Store the MTT solution away from light at 4 °C for repeated use or inC.20-° for long term storage2- Attenuation of visceral leishmania that lacks flagellum (responsible for penetration into the liver, spleen causing the infection and responsible for transmission, movement from one place to another) is the reason for the infection not occurring(Visceral leishmania was cultured after exposure in N199 medium and incubated at 25°C for four days.), Preparing a suspension using physiological salt solution and comparing its turbidity with the McFarland turbidity (NO.5) of attenuated visceral leishmania exposed to the Nd: YAG laser.3- attenuated Leishmaniadonovan y Nd: YAG laser and its medical application in Mice as attenuated vaccine in vivo) for immunization with elimination of visceral leishmaniasis( Withdraw 0.2 ml (200 pl) of the suspension and inject it into the mice into the peritoneum (12 mice) and compare it with a control with duplicates

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