الفهرس 
Historical backgroundOnly 14 pages are availabe for public view
 |  | from | 197 |  |  |
| | | from | 197 | | |
Abstract
The development of an effective vaccine against toxoplasmosis in humans remains a challenge to date. Extracellular vesicles (EVs) are nano-scale particles that are released from parasites and can induce efficient modulation of immunity, that makes them useful for probable next-generation vaccines.
Aim:
The study aimed to analyse whether exosomes (subtype of extracellular vesicles) purified from Toxoplasma gondii tachyzoites are able to immunize the mice and stimulate the protective immunity against challenge infection with T.gondii strains; highly virulent (RH) and avirulant (ME). The protective effectiveness was evaluated in comparison to excretory-secretory antigens (ESAs).
Methods:
Exosomes were purified from T. gondii tachyzoites using the Total Exosome Isolation Kit and then identified by TEM and western blot to be used for immunization of mice. The mice were randomly classified in various immunization groups; Exosomes-Alum, Exosomes, ESAs-Alum and ESAs and immunized with corresponding protein at days 1, 15, and 29 of the experiment. Using ELISA, the humoral and cellular immune responses were detected. After immunization, mice were challenged with either T. gondii RH or ME49 strains and the protective efficacy were assessed by recording the mortality daily and counting the brain cysts, respectively. Also, humoral and cellular immune responses were
assayed. Acute and chronic infected and non-infected mice were used as control groups.
Results:
Immunization of mice with Exosomes-Alum induced the highest level of T.gondii total IgG and cytokines (IFN-ℽ, IL-4, and IL-10) as well as CD8+T-lymphocytes count in the sera and splenocytes, respectively compared to ESAs-Alum. Mixed Th1/Th2 immune responses were induced in mice immunized with Exosomes-Alum. Exosomes-Alum immunization elongated the lifetime of mice challenged with virulant T. gondii strain to 11 days. It significantly reduced the brain cyst formation and evoked prominent cellular and humoral immune responses in mice challenged with avirulant T.gondii strain indicating a long duration maintenance of the protective effect compared to ESAs-Alum. Alum adjuvant boosts the efficacy of the Exosomes in inducing the protective immunity against T. gondii.
Conclusion:
The current study demonstrated that Exosomes-Alum was able to evoke strong anti-T.gondii immune responses and provide effective protection against T.gondii strains with different levels of virulence, suggesting that Exosomes-Alum may represent a promising immunization against toxoplasmosis.
ﻋﺎدةً ﻣﺎ ﺗﻜﻮن اﻟﻌﺪوى ﺑﻄﻔﯿﻞ اﻟﺘﻮﻛﺴﻮﺑﻼزﻣﺎ ﺟﻮﻧﺪاى ﻏﯿﺮ ﻣﺼﺤﻮﺑﮫ ﺑﺄﻋﺮاض ﻓﻲ اﻷﻓﺮاد اﻟﻤﺆھﻠﯿﻦ ﻣﻨﺎﻋﯿﺎ، ﻏﯿﺮ أﻧﮫ ﻓﻰ اﻷﻓﺮاد اﻟﻤﺜﺒﻄﯿﻦ اﻟﻤﻨﺎﻋﺔ ﯾﺤﺪث ﺗﻨﺸﯿﻂ ﻟﻸﻛﯿﺎس اﻟﻄﻔﯿﻠﯿﮫ ﺑﺎﻟﻤﺦ
ﻛﻤﺎ أﻧﮫ ھﻨﺎك اﺣﺘﻤﺎل
اﻧﺘﺸﺎر اﻟﻌﺪوى،
ﻓﺘﺆدي إﻟﻰ اﻹﺻﺎﺑﺔ ﺑﺪاء اﻟﻤﻘﻮﺳﺎت اﻟﺪﻣﺎﻏﻲ أو
ﻛﺒﯿﺮﻻﻧﺘﻘﺎل اﻟﻌﺪوى اﻟﻄﻔﯿﻠﯿﮫ ﻣﻦ اﻻم اﻟﺤﺎﻣﻞ اﻟﻰ ﺟﻨﯿﻨﮭﺎ ، وﻟﺴﻮء اﻟﺤﻆ ﻓﺈن اﻟﻌﻼج اﻟﺪواﺋﻲ
اﻟﻤﻘﻮﺳﺎت
ﻋﻠﻰ ﻗﺘﻞ
ﻗﺪرﺗﮫ
ﻧﮭﺎﺋﯿﺎ ﻟﻌﺪم
ﻋﻠﻰ داء اﻟﻤﻘﻮﺳﺎت
ﯾﻘﻀﻲ
ﻻ ﯾﺴﺘﻄﯿﻊ أن
اﻟﺤﺎﻟﻲ
اﻟﻤﺘﺒﺎطﺌﺔ (Bradyzoites) ، ﻟﺬﻟﻚ ﺗﻘﺪم اﻟﻠﻘﺎﺣﺎت ﺑﺪﯾﻼ أﻓﻀﻞ ﻟﻠﺴﯿﻄﺮة اﻟﻔﻌﺎﻟﺔ ﻋﻠﻰ اﻟﻤﺮض
ﻋﻠﻰ اﻟﻤﺪى اﻟﻄﻮﯾﻞ.
ان أﺳﺘﺨﺪام اﻟﺤﻮﯾﺼﻼت اﻟﺨﺎرج ﺧﻠﻮﯾﺔ اﻟﻤﺸﺘﻘﺔ ﻣﻦ اﻟﺘﻜﺴﻮﺑﻼزﻣﺎ ﺑﺄﻧﻮاﻋﮭﺎ اﻟﻔﺮﻋﯿﺔ اﻟﺜﻼﺛﺔ )اﻟﺤﻮﯾﺼﻼت اﻟﺪﻗﯿﻘﺔ واﻹﻛﺴﻮﺳﻮﻣﺎت واﺟﺴﺎم ﻣﻮت اﻟﺨﻼﯾﺎ اﻟﻤﺒﺮﻣﺞ( ﻛﻠﻘﺎح أﺛﺎرت اﻻﻧﺘﺒﺎه
اﻟﺪراﺳﺔ اﻟﺤﺎﻟﯿﺔ إﻟﻰ ﻋﺰل وﺗﻮﺻﯿﻒ
اﻷﺟﯿﺎل اﻟﻘﺎدﻣﮫ. ھﺪﻓﺖ
ﻟﻘﺎح
ﻻﺣﺘﻤﺎﻟﯿﮫ ﻛﻮﻧﮭﺎ
ﺛﻢ اﺳﺘﺨﺪاﻣﮭﺎ ﻛﻠﻘﺎح ﻣﻨﻔﺮد أو
ﺟﻮﻧﺪاى
اﻟﺘﻜﺴﻮﺑﻼزﻣﺎ
طﻔﯿﻞ
اﻟﻤﺸﺘﻘﺔ ﻣﻦ
اﻹﻛﺴﻮﺳﻮﻣﺎت
ﺑﺎﻟﻤﻘﺎرﻧﺔ ﻣﻊ
ﻓﻰ اﻟﻔﺌﺮان
ﺿﺪ داء اﻟﻤﻘﻮﺳﺎت اﻟﺘﺠﺮﯾﺒﻲ
ﻣﺤﻔﺰة(Alum)
ﻣﺼﺤﻮﺑﺎ ﺑﻤﺎدة
ﻣﺴﺘﻀﺪ ESAs
ﺗﻢ ﺗﻤﺮﯾﺮ اﻟﺴﻼﻟﺔ ﺷﺪﯾﺪة اﻟﻀﺮاوة ﻣﻦ اﻟﻤﻘﻮﺳﺎت اﻟﻤﺘﺴﺎرﻋﺔ (Tachyzoites) ﻓﻲ اﻟﺘﺠﻮﯾﻒ
اﻛﺴﻮﺳﻮﻣﺎت
ﺗﻢ ﻋﺰل
Kit
وﺑﺎﺳﺘﺨﺪام
. Hep G2
اﻟﺒﺮﯾﺘﻮﻧﻲ ﻟﻠﻔﺌﺮان ﺛﻢ زراﻋﺘﮭﺎ ﻋﻠﻰ
اﻟﺘﻜﺴﻮﺑﻼزﻣﺎ ﺟﻮﻧﺪاى ﻣﻦ اﻟﻤﻘﻮﺳﺎت اﻟﻤﺰروﻋﺔ ﺛﻢ ﺗﻮﺻﯿﻔﮭﺎ ﺑﺎﺳﺘﺨﺪام اﻟﻤﺠﮭﺮ اﻻﻟﻜﺘﺮوﻧﻲ
وﺗﺤﻠﯿﻞ اﻟﻠﻄﺨﮫ اﻟﻤﻨﺎﻋﻲ blot) (Western . أظﮭﺮ اﻟﺘﻮﺻﯿﻒ ﺑﻮاﺳﻄﺔ اﻟﻤﺠﮭﺮ اﻻﻟﻜﺘﺮوﻧﻲ أن اﻟﺤﻮﯾﺼﻼت ﻛﺎﻧﺖ ﻛﺮوﯾﺔ اﻟﺸﻜﻞ وﺑﺄﻗﻄﺎر ﺗﺘﺮاوح ﺑﯿﻦ 150-50) ﻧﺎﻧﻮﻣﺘﺮ( ، ﻛﻤﺎ أﺛﺒﺖ ﺗﺤﻠﯿﻞ اﻟﻠﻄﺨﮫ اﻟﻤﻨﺎﻋﻲ blot) (Western إﯾﺠﺎﺑﯿﺘﮭﺎ ﻟـ CD63 وCD81 ، وھﻤﺎ دﻻﺋﻞ ﻓﺮﯾﺪة
ﻟـﻼﻛﺴﻮﺳﻮﻣﺎت.
ﺗﻢ اﺳﺘﺨﺪام اﻻﻛﺴﻮﺳﻮﻣﺎت اﻟﻤﺸﺘﻘﺔ ﻣﻦ اﻟﺘﻜﺴﻮﺑﻼزﻣﺎ ﺟﻮﻧﺪاى ﻓﻲ ﺗﺤﺼﯿﻦ اﻟﻔﺌﺮان ﻣﻊ وﺑﺪون
ﻣﺎده ﻣﺤﻔﺰة Alum ﻣﻘﺎرﻧﺔً ﺑﻤﺴﺘﻀﺪ ESAs ، ﺗﻢ ﺗﺤﺼﯿﻦ اﻟﻔﺌﺮان ﻓﻲ اﻷﯾﺎم 1 و 15 و 29 ﻣﻦ اﻟﺘﺠﺮﺑﺔ. ﺛﻢ اﻟﺤﺼﻮل ﻋﻠﻰ ﻋﯿﻨﺎت اﻟﺪم ﻋﻦ طﺮﯾﻖ ﺛﻘﺐ اﻟﻀﻔﯿﺮة اﻟﻤﺪارﯾﺔ ﻓﻲ اﻟﻌﯿﻦ ﻓﻲ اﻷﯾﺎم 0 و 14 و 28 و 56 ﻣﻦ اﻟﺘﺠﺮﺑﺔ ﺑﯿﻨﻤﺎ ﺗﻢ اﻟﺤﺼﻮل ﻋﻠﻰ ﻋﯿﻨﺎت ﻣﻦ اﻟﻄﺤﺎل ﻓﻲ اﻟﯿﻮم
ﺗﻢ ﻗﯿﺎس إﺟﻤﺎﻟﻲ اﻷﺟﺴﺎم اﻟﻤﻀﺎدةIgG ﺿﺪ
ELISA
و ﺑﺎﺳﺘﺨﺪام
56 ﻣﻦ اﻟﺘﺠﺮﺑﺔ.
اﻟﺘﻮﻛﺴﻮﺑﻼزﻣﺎ ﺟﻮﻧﺪاى ﻓﻲ ﺳﯿﺮم اﻟﺪم و IFN-ℽ و IL-4 و IL-10 ﺑﺎﻹﺿﺎﻓﺔ إﻟﻰ ﻋﺪد ﺧﻼﯾﺎ
CD4+و CD8+ ﻓﻲ اﻟﺨﻼﯾﺎ اﻟﻄﺤﺎﻟﯿﺔ.
وﻟﺘﻘﯿﯿﻢ اﻟﻔﻌﺎﻟﯿﺔ اﻟﻮﻗﺎﺋﯿﺔ ﻟﻠﻘﺎح ﺗﻤﺖ ﻋﺪوى اﻟﻔﺌﺮان اﻟﻤﺤﺼﻨﺔ ﻓﻲ اﻟﯿﻮم 60 ﻣﻦ اﻟﺘﺠﺮﺑﺔ إﻣﺎ
طﺮﯾﻖ اﻟﻔﻢ
أو ﻋﻦ
اﻟﻤﻘﻮﺳﺎت اﻟﻤﺘﺴﺎرﻋﺔ
ﻟﻠﺴﻼﻟﺔ ﺷﺪﯾﺪة اﻟﻀﺮاوة ﻣﻦ
ﺑﺎﻟﺤﻘﻦ اﻟﺒﺮﯾﺘﻮﻧﻲ
ﺑﺎﻷﻛﯿﺎس اﻟﻤﺘﺠﺎﻧﺴﺔ اﻟﺪﻣﺎﻏﯿﺔ ﻟﻠﺴﻼﻟﺔ اﻷﻗﻞ ﺿﺮاوة. ﺗﻤﺖ ﻣﺮاﻗﺒﮫ اﻟﻔﺌﺮان اﻟﻤﻌﺪﯾﮫ ﺑﺎﻟﺴﻼﻟﺔ
اﻟﺸﺪﯾﺪة ﺿﺮاوة ﯾﻮﻣﯿًﺎ ﻟﻤﻌﺮﻓﮫ ﻣﺪة ﺑﻘﺎءھﺎ ﻋﻠﻰ ﻗﯿﺪ اﻟﺤﯿﺎة. ﻛﻤﺎ ذﺑﺤﺖ اﻟﻔﺌﺮان اﻟﻤﻌﺪﯾﮫ ﺑﺎﻟﺴﻼﻟﺔ اﻷﻗﻞ ﺿﺮاوة ﻓﻲ اﻟﯿﻮم 60 ﺑﻌﺪ اﻟﻌﺪوى ﻟﻌﺪ أﻛﯿﺎس اﻟﻄﻔﯿﻞ ﻓﻲ اﻟﻤﺦ ﻛﻤﺎ ﺗﻢ ﺗﻘﯿﯿﻢ اﻻﺳﺘﺠﺎﺑﺎت
اﻟﻤﻨﺎﻋﯿﺔ اﻟﺨﻠﻄﯿﺔ واﻟﺨﻠﻮﯾﺔ وﺗﻢ ﺗﺤﻠﯿﻞ اﻟﻨﺘﺎﺋﺞ إﺣﺼﺎﺋﯿﺎ.
اﻟﻤﺤﻔﺰة
ﻣﺼﺤﻮﺑﮫ ﺑﺎﻟﻤﺎدة
اﻟﻔﺌﺮان اﻟﻤﺤﺼﻨﺔ ﺑﺎﺳﺘﺨﺪام اﻹﻛﺴﻮﺳﻮﻣﺎت
أظﮭﺮت اﻟﺪراﺳﺔ أن
(Exosomes-Alum)ﻗﺪ ﺣﻘﻘﺖ أﻋﻠﻰ ﻣﺴﺘﻮﯾﺎت ﻣﻦ اﻷﺟﺴﺎم اﻟﻤﻀﺎدة ﻟﻄﻔﯿﻞ اﻟﺘﻮﻛﺴﻮﺑﺎزﻣﺎ ﺟﻮﻧﺪاى IgG اﻟﺘﻲ ﺗﺰداد ﺗﺘﺎﺑﻌًﺎ ﻣﻊ ﺗﻌﺎﻗﺐ ﺟﺮﻋﺎت اﻟﺘﺤﺼﯿﻦ وﻛﺬﻟﻚ ﺣﻘﻘﺖ أﻗﻮى اﺳﺘﺠﺎﺑﺔ
ﻣﻨﺎﻋﯿﺔ ﺧﻠﻮﯾﺔ ﻣﻘﺎرﻧﺔ ﺑﺘﻠﻚ اﻟﻤﺤﺼﻨﺔ ﺑﺎﻟﻤﺴﺘﻀﺪ ﻣﺼﺤﻮﺑﺎ ﺑﺎﻟﻤﺎده اﻟﻤﺤﻔﺰة ESAs-Alum .
ﻛﻤﺎ أن ال Exosomes-Alumﻗﺪ ﺣﻘﻘﺖ زﯾﺎده ﻣﻠﺤﻮظﮫ اﺣﺼﺎﺋﯿﺎ ﻓﻰ ﻣﺴﺘﻮﯾﺎت اﻟﺴﯿﺘﻮﻛﯿﻨﺎت
IFN-ℽ)و IL-4 و (IL-10 وﻋﺪد ﺧﻼﯾﺎ CD8+ ﻓﻲ اﻟﻔﺌﺮان.
ﺑﻌﺪ اﻟﻌﺪوى ﺑﺎﻟﺴﻼﻟﮫ ﺷﺪﯾﺪة اﻟﻀﺮاوة، أﺳﺘﻤﺮت اﻟﻔﺌﺮان اﻟﻤﺤﺼﻨﺔ ﺑـ Exosomes-Alum
ﻋﻠﻰ ﻗﯿﺪ اﻟﺤﯿﺎه وﻗﺘًﺎ أطﻮل 11) ﯾﻮﻣﺎ( ﻣﻘﺎرﻧﮫ ﺑﺎﻟﻔﺌﺮان اﻟﻤﺤﺼﻨﺔ ﺑـ ESAs-Alum 9) أﯾﺎم.( ﺑﯿﻨﻤﺎ ﺑﻌﺪ 60 ﯾﻮﻣﺎ ﻣﻦ اﻟﻌﺪوى ﺑﺎﻟﺴﻼﻟﮫ اﻻﻗﻞ ﺿﺮاوة، اﻧﺨﻔﺾ ﻋﺪد أﻛﯿﺎس اﻟﻄﻔﯿﻞ ﺑﻤﺦ اﻟﻔﺌﺮان اﻟﻤﺤﺼﻨﺔ ﺑـ Exosomes-Alum ﺑﺸﻜﻞ ﻛﺒﯿﺮ ﻣﻘﺎرﻧﺔ ﺑﺘﻠﻚ اﻟﻤﻮﺟﻮدة ﻓﻲ اﻟﻔﺌﺮان اﻟﻤﺤﺼﻨﺔ
42 ٪ ﻓﻘﻂ ﻓﻲ اﻟﻔﺌﺮان
ﻣﻘﺎرﻧﺔ ﺑـ
ﻣﻌﺪل اﻻﻧﺨﻔﺎض إﻟﻰ 75
ESAs-Alumﺣﯿﺚ وﺻﻞ
اﻟﻤﺤﺼﻨﺔ بESAs-Alum .
ﻛﻤﺎ أظﮭﺮت ﻣﺴﺘﻮﯾﺎت اﻷﺟﺴﺎم اﻟﻤﻀﺎدة IgG وﺟﻤﯿﻊ اﻟﺴﯿﺘﻮﻛﯿﻨﺎت وﺧﻼﯾﺎ CD4+ و CD8+ زﯾﺎدة ﻣﻠﺤﻮظﮫ اﺣﺼﺎﺋﯿﺎ ﻓﻲ اﻟﻔﺌﺮان اﻟﻤﺤﺼﻨﺔ ﺑﺎل Exosomes-Alum ﻣﻘﺎرﻧﺔ ﺑﻤﺴﺘﻮﯾﺎﺗﮭﺎ ﻓﻲ ﻣﺠﻤﻮﻋﺎت اﻟﺘﺤﺼﯿﻦ ESAs ، ﻣﻤﺎ ﯾﺸﯿﺮ إﻟﻰ أن اﻟﻔﻌﺎﻟﯿﮫ اﻟﻮﻗﺎﺋﯿﮫ ﻟﻠﻘﺎح اﻻﻛﺴﻮﺳﻮﻣﺎت ﻛﺎﻧﺖ طﻮﯾﻠﮫ اﻷﻣﺪ. ﺑﺎﻻﺿﺎﻓﺔ إﻟﻰ ذﻟﻚ ، وﺟﺪ أن اﺿﺎﻓﮫ اﻟﻤﺎده اﻟﻤﺤﻔﺰة ﻟﻠﻘﺎح اﻻﻛﺴﻮﺳﻮم ﻛﺎن
ﻟﮭﺎ أﺛﺮا ﻛﺒﯿﺮا ﻓﻰ زﯾﺎده ﻓﻌﺎﻟﯿﮫ ﻟﻘﺎح اﻻﻛﺴﻮﺳﻮم.
اﻟﻤﻘﻮﺳﺎت
اﻟﻤﻌﺰوﻟﺔ ﻣﻦ
Exosomesال أن
و ﻣﻦ ﻛﻞ ﻣﺎ ﺳﺒﻖ, ﻓﻘﺪ ﺧﻠﺼﺖ اﻟﺪراﺳﺔ إﻟﻰ
اﻟﻤﺘﺴﺎرﻋﺔ ﻟﻄﻔﯿﻞ اﻟﺘﻮﻛﺴﻮﺑﻼزﻣﺎ ﺟﻮﻧﺪاى واﻟﺘﻲ اﺳﺘﺨﺪﻣﺖ ﻣﺼﺤﻮﺑﮫ ﺑﺎﻟﻤﺎده اﻟﻤﺤﻔﺰة Alum ﻓﻲ ﺗﺤﺼﯿﻦ اﻟﻔﺌﺮان ﻗﺪ أدت إﻟﻰ اﺳﺘﺠﺎﺑﺎت ﻣﻨﺎﻋﯿﺔ ﺧﻠﻄﯿﺔ وﺧﻠﻮﯾﺔ ﻗﻮﯾﺔ وﺣﻤﺎﯾﺔ ﺟﺰﺋﯿﺔ ﺿﺪ ﻛﻼ ﻣﻦ اﻟﻌﺪوى اﻟﺘﺠﺮﯾﺒﯿﮫ اﻟﺤﺎدة واﻟﻤﺰﻣﻨﺔ ﻟﺪاء اﻟﻤﻘﻮﺳﺎت و اﻟﺘﻰ اﺗﻀﺤﺖ ﻓﻰ ﺑﻘﺎء اﻟﻔﺌﺮان
ﻋﻠﻰ ﻗﯿﺪ اﻟﺤﯿﺎه ﻟﻔﺘﺮة أطﻮل وﻧﻘﺺ ﻣﻠﺤﻮظ ﻓﻰ ﻋﺪد أﻛﯿﺎس اﻟﻄﻔﯿﻞ ﺑﺎﻟﻤﺦ ، ﻋﻠﻰ اﻟﺘﻮاﻟﻲ، ﻣﻘﺎرﻧﺔً
ﺑﺘﺤﺼﯿﻦESAs . ﻛﻤﺎ ان أﺿﺎﻓﮫ اﻟﻤﺎدة اﻟﻤﺤﻔﺰة ﻟﻠﻘﺎح Exosomes ﻛﺎن ﻟﮫ أﺛﺮ ﻛﺒﯿﺮ ﻓﻰ
ﺗﻌﺰﯾﺰ اﻟﻔﻌﺎﻟﯿﮫ. و ﻟﺬﻟﻚ ﻓﺈن اﻟﻨﺘﺎﺋﺞ اﻟﺤﺎﻟﯿﺔ ﺗﺸﯿﺮ إﻟﻰ أن Exosomes ﺑﺎﻻﺷﺘﺮاك ﻣﻊ Alum
ﻗﺪ ﺗﻜﻮن ﺑﻤﺜﺎﺑﺔ ﻣﺮﺷﺢ ﻣﺤﺘﻤﻞ ﻛﻠﻘﺎح ﺿﺪ داء اﻟﻤﻘﻮﺳﺎت