Parte 3 — Jacques Tremblay — Seminário Internacional de Ciências Farmacêuticas — Método CRISPR

Análise Clínica e Orientação Médica sobre Parte 3

Thank you for the invitation let me start my presentation using the crisper cast 9 technology today's presentation will be divided into part i will first present the use of the crispr cas9 technology to treat iris daily disease and my graduate student gabriel de mart will present all the crispr cas9 derived technology may be used to detect viral infection the word crispr stands for clustered regulatory interspace short penetrate repeats crispr cas9 technology is derived from research on bacteria it was initially discovered in 2005 that the crisper sequence contained viral sequence and yes there was an hypothesis that crispr cas9 is the bacterial immune system it's only in 2013 that it was discovered that the crispr cas9 may be used to induce specific modification of the human genome and this started a big explosion of artificial indeed the crispr casting system is used by bacteria to kill bacteriophage bacteriophages are extremely abundant they're 10 times more abundant than bacteria and there is a war that is ongoing between bacteria and bacteriophage since millions of years it was initially discovered that they are in the genome a constant repeated sequence researchers were wondering what these repeated sequences were for and then they discovered that the sequence between the constant repeat or sequence that were up from the genome of various bacteriophage in fact what normally happens is when a bacteriophage infected bacteria normally the bacteriophage wind bacteria is dead but sometimes you have a defective virus that will infect without killing the bacterium but they will then be able to acquire some part of the sequence of the virus and it will store that into its genome and it will be ready when it needs such bacteriophage again in the future from the dna in the crispr area bacteria will first express the pre-cr allergy and then there will be a complex that will be made between tracker or cr rna and the cast iron protein this striker rna crna cas9 protein complex will then bind with the bacteria of hdna and induce a cut of the bacteriophage dna to essentially kill the virus to bind to the dna the cas9 protein requires the presence of the total spacer adjacent motif which is simply njj for this streptococcus pyrogene then there is a crrna which contain a variable sequence of 20 nucleotides that will be complementary to the bacteriophage sequence and then there is also the tracker rna which is a constant rna sequence that forms a complex with the crm when the three parts are together cr rna tracker rna in the cas9 protein the cut will be induced at exactly three nucleotides of course since this is a war there are a counter-measure that are taken by the phage they will mutate their genome so that they become resist and they are not recognized by the cr rna and will survive bacterial defense the crispr cas9 big bang started in 2012 when gen x and colleagues realized that the crispr castling technology can permit to cut the genome of planets and animals they also fuse their cr rna and the tracker rna together to form a single guide rna this was done essentially because the single guide rna is something that can be patented whereas the cr rna and the tracker have any being natural it cannot be patented during the following year there was an explosion of article confirming that the crispr technology may be used to cut the genome of bacteria human cells carb animal mouse frogs zebrafish plant flights limited these monkeys many of these articles were published in prestigious journals such as nature science nature biotechnology molecular cell molecular cell there was a rapid explosion of article and as you can see as of november 26 2020 21 420 articles have been published where the word crispr is mentioned the main reason of this rapid explosion of article is that it costs much less to derive a sequence that will be able to cut a specific genome sequence compared to other pre-existing technology such as zinc finger protein and tailors as mentioned before the binding of the cas9 protein to the dna required the presence of a bottle spacer adjacent concept pam which is njj for the cas9 derived from skeptical g there is then the binding also of the single guide rna which will recognize the sequence of 20 nucleotides and when the complex is made between the cas9 protein the single guide rna the dna there is a cut that will be made at exactly three nucleotides from the pan once inside the nucleus the resulting complex will lock onto a short sequence known as the pan the cas9 will unzip the dna and match it to its target rna if the match is complete the cast 9 will use two tiny molecular scissors to cut the dna when this happens the cell tries to repair the cut but the repair process is error-prone leading to mutations that can disable the gene allowing researchers to understand its function these mutations are random but sometimes researchers need to be more precise for example by replacing a mutant gene with a healthy copy this can be done by adding another piece of dna that carries the desired sequence once the crispr system has made a cut this dna template can pair up with the cut ends recombining and replacing the original sequence with the new version one of the reasons of the rapid explosion is that increased bypass like technology also used all what was developed by gene therapy in the 30 years that preceded it we have better to delivering the dna using aav associated virus cationic polymers liposome microinjection electro operation all of those techniques are used in the context of crispr when a double strand break is produced in the dna most of the breaks will be repaired by non-homologous adjoining that will lead to micro deletion or micro insertion this is frequently used to knock out the expression of a gene by changing the reading frame and it can also be repaired by homology directed repair which require the presence of a donor dna that contains sequence of homology with what is receding what is following the cut and in between the two homology sequence there is a sequence in blue which may be a single nucleotide or a whole gene that will be inserted at the side of the double strand break when we use the crispr cas9 technology we intend to produce a double strand break at a precise site in the human genome however the cas9 may sometimes induce cut at other sites in the genome which are called off-target mutation computer software may predict these off-target mutation by using the whole human genome sequence however these softwares are not perfect and they will sometimes print it off-target mutation at site where there is no cut and sometimes there will be a cut at the side which is not predicted by the computer there are or however some experimental techniques such as a guide sec method which permit to experimentally identify the sites of target mutation this guidesec technique used the introduction of a short 34 base pair oligonucleotide at the site of the cuts and later by pcr the site of insertion may be identified by sequencing the presence of these off-target mutations is the main problem that delays the use of crispr cat9 technology for direct vehicle corrections one of the method to reduce of target mutation is simply to reduce by two or three the number of nucleotides of the single guide rna which is binding with the target dna another method is to use a mutated cas9 nuclease that cut only one strand of dna it is then called the kneecase there is variation of cas9 where the nikkei cut only the lower strand of dna and other mutation of cas9 which permit to cut only the upper strand of dna it is just possible to induce more precise double strand break in the dna by using the cas9 nikkei and two single guide rna each detecting a sequence of 20 nucleotides close to one another it is also possible to use a non-functional cast 9 that then cast 9 nuclease fuse with the one nucleates then two guide iron area needed to detect sequence which are close to another and the front one nucleas need to form a dimer in order to be able to cut the dna finally some researchers have mutated the gene coding from cas9 they have modified some sequence coding for amino acid to reduce the non-specific binding between the cas9 and the dna the sp cas9 gene is too big to be inserted with a single guide rna inside an adeno associated virus and thus researchers have identified cas9 from other bacteria that are smaller for example this staphylococcus probably is cast 9 it is smaller than the sp 9 but it has a different path which is more restrictive another type of crispr cas9 enzyme has been identified it's called cpf1 it produces sticky cancers it's another one cut of the dna there is just a whole series of various castings obtained from various bacteria that require different types the crisprcas9 technology may be used not only to reduce cuts in the dna but it may also be used to induce the expression of a gene by fusing cast 9 with bp 64 for example or iron with crab to repress the expression of a gene in my research group we are using the crispr cas9 technology to develop therapies for various disease much ataxia design muscular dystrophy and alzheimer's disease i will present that now we have initially used the crispr cas9 technology to develop a treatment for freshwater taxi it is an adidas disease due to the presence of a long tri-nucleotide with bgaa in intron one of the fertilizer gene this long repeat reduce the expression of the fataxin gene leading to neurological problems and cardiac problems we have just used the crispr cas9 technology and generated single guide rna able to cut in intron 1 before and after the trinucleotide repeat leading to its removal and increasing the expression of ataxia indeed the removal of the tri-nucleotide repeat in cells of the yg8 sr mouse model of frederic ataxia doubled the expression of fataxin compared to the untreated cells and raise the expression of protection to almost the normal level the sp cas9 gene is too big to be delivered with two single gut rna by a single aav we are just currently using the smaller cgcas9 gene to remove the trinucleotide in intron one of the fataxin gene using this smaller cg cas9 nuclease we are also able to remove the trinucleotide repeat in intron 1 of the fratexin gene we are also using the crispr cas9 technology to develop a treatment for duchenne muscular dystrophy duchenne muscular dystrophy is due to mutation in a gene coding for the dystrophin protein it is a large gene containing 79 exons some of these exons do not contain a multiple of three nucleotides and thus seventy percent of duchenne muslim dystrophy cases are due to deletion of one or several exon and the total number of coding nucleotides which are deleted is not a multiple of three for example in this presentation we have a deletion of exon 50 that does not contain a multiple of three nucleotides this lead to a frame shift and there will be a stop codon in exon 51.

Thus the beginning of this the dystrophin protein is expressed but not the end of the dystrophin protein this situation may be corrected by inducing the skipping that is the removal of exon 51 in the messenger rna this is done using antisense oligonucleotide this exon 51 may also be deleted by inducing cuts in intron 50 and in front 51 to completely remove that exon this results in the expression of the beginning of the dystrophin protein and of the end of the dystrophin protein but there is however a small part of the protein which is missing in the center of the protein the removal of one or several complete exam may thus restore the reading frame and convert a duchene patient into a becker patient however some backer patients have severe symptoms and are bound to a wheelchair at the age of 11.

Therefore the improvement of some duchenne muscular dystrophy may not be significant this is because the dystrophin protein has a complex structure indeed in its central part the dystrophin protein contains 24 spectrum-like repeats each spectrum ligatude is made of three alpha helix a linux b and exc note that nx a is starting on the left side and elix c is ending on the right side and normally there is a succession of abc the main problem of deleting complete exam to restore the expression of dystrophin protein is that the beginning and the end of the spectrum-like repeat indicated in this scheme by the black arrow do not correspond with the beginning and the end of exiles and thus when removing complete absorbs the resulting of the spectrum-like repeat structure may not be normal vector muscularis computation have a deletion of one or several exon but the total number of coding nucleotides which are deleted is a multiple of three nucleotides and thus there is no frame shift this is the case for example for becker patient having a deletion of example 45 to 47 or having a deletion of 45 to 49 in both case there's no frame shift the beginning and the end of the dystrophin protein are expressed however at the junction site between the remaining codons there is an abnormal structure of the dystrophin protein and due to this abnormal structure of the dystrophin protein these vector patients are bound to a wheelchair at an early age thus in my research group instead of trying to restore the normal reading frame by deleting complete exons we have instead aim at producing an evaline exam 5054 which not only restore the normal reading frame but calls for a dystopian protein with a normal structure we have done our initial experiments using the myoblast of a duchenne patient having a deletion of exam 51 52 and 53.

Thus because the number of coding nucleotides was not a multiple of three there was a stop codon in exam 54 due to the frame shift we have just used a crisper cas9 technology to induce a cut in exon 50 and a cut in exon 54 to create the e-braid exam 50-54 to produce this immigration exam we initially identify what are the possible sp cas9 pam or what other site where we can cut in exon 50 and in exon 54 we identify 10 time sites in exam 50 and 14 different pan size in exon 53.

This table predicts what are the results of inducing cuts in exam 50 and in exam 54. When we have a blue square it means that the cut has been produced in exon 50 right after the three nucleotides and calls for an amino acid and the cut has been produced in exon 54 just before the three nucleotide that codes for an amino acid thus at the junction point we have amino acids that are one escorted by example 54 followed immediately by an amino acid coded by example there's no friendship and there is no new amino acid produced however when the square is clean that means that we have at the junction site a new amino acid which is produced because we are cut in exon 50 right after one of the nucleotides of the codon and the two nucleotides that will complete these codons are from exotic before we have cut right after one nucleotide of the codon in exam 54 and thus first two nucleotides left to create a new codon at the junction site there's just a new amino acid at the junction site but there is no frame shift and all the other amino acids are the correct ones it is also possible with this square in red to have a new codon produced at the junction side but this new codon is a stop codon this is not real we want to do all the squares in wide because there is a frame shift we're cutting after one nucleotide of the codon in exon 15 and we're cutting before the last nucleotide of the cooldown in exam 54 and thus add the junction side there is a new codon which is done because there is a frame shift and all the amino acids that follows are not the correct one we have dust testers there your sphere of guide irony one guide irony cutting in zone 50 and the other guide ironing cutting in exon 54 this produced an ibrit exam 1554 that had the predicted size of this hybrid exam note that when we are doing cuts in exam 50 and in exon 54 in a normal dystrophin g we are deleting 160 000 base pair and despite that the junction between exon 50 and 54 is of the size predicted we have sequence this elite exam that was produced by cutting in exon 50 and in exon 54.

Not only were the eblid exam exactly the size as predicted but the sequence were also exactly as predicted for example we had a junction site the fusions of exon 50 and example 54 producing exactly the predicted amino acid we also obtained at the junction site a new codon coding for the predicted amino acid or coding for a stop codon and sometime as predicted by the white square we had a frame shift and when there was a frame shift there were stub cooldowns that were met in the resulting exams as mentioned before the dystrophin protein contained in its central part 24 spectrum like repeat each one being made of three alpha helix a b and c we have just used a guide rna to induce a cut in exon 50 in a sequence coding for lxc and another guide rna inducing a cut in exon 54 also in a sequence coding for helix c the resulting evidence exon 5054 calls for an ibrahim alex c where the beginning of nxt is coded by exon 50 and the end of lxc is coded by exon 54.

This is a structure that has been computer predicted using the sequence of the resulting protein when the bioglass of this duchenne patient having a deletion of exam 51 to 53 are fused together to form some small muscle fibers and culture called myotubes in fact these muscle fibers do not express dystrophin because this is a shame patient however the myoblast of a healthy human when they fuse together they form a small biotube and they do express this topic as can be seen here in this western blood when we use the bioblast of the duchenne patient and we induce the formation of the ignition exam 50-54 we have expression of the dystrophin protein in culture and as you can see this dystrophin protein has a lower molecular weight than the normal dystrophin because there is deletion of exon 51 to 53 and an additional deletion of part of exon 50 part of the exam 54 thus there is a part of the dystrophin protein which is absent and this is why the protein is smaller but it is nevertheless expressed for our initial in vivo experiments we have used the hdmd mouse model this is a transgenic mouse that expressed the human dystrophin gene all introns in all exons we have electroporated in the muscle of this mouse plasmid coating for the sp cas9 fuels with the fluorescent green protein and two single guide rnas this a month later when we took the muscle there was expression of the green fluorescent protein confirming that the plasmid had been correctly electroporated in the muscle fibers leading to the expression of the fluorescent green protein and probably also leading to the expression of the sp cast iron and after two glide ironing we then extracted the dna from these muscle and we first confirmed using a test called the surveyor enzyme that indeed they were constantly being produced in exam 50 and 54.

We then use pcr to amplify the ebrade exon 5054 note that in the muscles that are not treated with the crispr cas9 technology there is no amplification of example 5054 because there are 160 000 base pair between these two exons and therefore the two primers for the pcr are too much separated from one another we then sequence this exon 5054 that was produced in vivo in the mouse muscle expressing the human dystrophin gene and as predicted we had the sequence of exon 50 and a new codon at the junction site followed by all the correct codon coding for the normal amino acid in exon 54.

This is exactly as predicted in vivo however for a delivery of the cas9 gene in vivo to several muscle we need to use an adeno associated virus as mentioned before the sp cas9 g is too big to be delivered with two guide irony by a single aav and we therefore use for our next experiments the cast 9 of the staphylococcus moleus which is a smaller cas9 which permit delivery with two guide rna by a single avp in this case we have used a guide rna that conducts cuts in exon 47 in a sequence coding for elix b and in exon 58 in a sequence again coding for elix b this resulted in the formation of an ebola exome 4758 coding for an ibrigid hxb which has a normal structure the beginning of the lxb being coded by exon 47 and the end of that evil be encoded by exon 58 we then use for our indivo experiment a new mouse model called hdmp delta ii it's the same mouse model as previously that contains the dystrophin gene with all the exons and all the intron except that this mouse has a deletion of exam 52 and thus there is no expression of the human dystrophing gene we have thus used the crispr cas9 technology to induce a cut in exon 47 and a cut in exam 58 producing the eberlini exam 4758 resulting in the production of a dystopian protein containing an ibrite example in lxp we have injected to these hdmd delta 52 mouse aev's coding for sc cast 9 and 2 single guide rna a month later we observe in the muscle the expression of the human dystrophin gene including in the heart we are just proposing a treatment of duchenne muscular dystrophy which would be the systemic delivery by an adeno associated virus of the class 9g and of two guide rna to form any bread exam in contrast with exam skipping which is a treatment done at the level of the message rna the treatment that we propose at the level of the dna would be permanent the crispr cas9 technology is evolving rapidly and new technologies derived from the crispr cas9 permits now the modification of a single nucleotide more than 32 000 single nucleotide modifications are responsible for hearing steady disease and thus the capacity to correct a single nucleotide would provide treatment for most of these healing steady disease the first treatment which permit the modification of a single nucleotide was developed by command in 2017 which used a cas9 knee case that is the modified class cas9 which is able to cut only one strand of dna and this cast 90 case is fused with acetidine deaminase this technology impermanently clinically modify city bean into a uvd which is replaced by a timing of dna repair the main limitation of that technique is the chemical modification of the ctd will occur in a narrow window located at 12 to 16 base pair from the ngg pan we have initially used base editing technology to develop a treatment for alzheimer's disease the alzheimer's disease is produced by an abnormal metabolism of the ammunition protein normally this protein is cut by the alpha secretaries followed by a cut by the gamma symmetries this produce peptides and protein fragments that are degraded without causing any problem however this protein may also be cut by the beta sequence followed by a cut by the gamma secretaries and this produced short 40 42 amino acid long beta middling peptides that aggregates to one another forming amino acids that interfere with synaptic transmission leading to neuron death and the memory problems this scheme illustrates the amino acid sequence of the transmembrane part of the anaerobic repressure protein we can see the position of the beta alpha and gamma separates outside all the amino acids the star above their name are amino acids which are quantified leading to family of form version of disease please note the position with the red arrow this is the alanine in version 673 when this adenine is changed by valley this leads to severe early onset alzheimer's disease and you are azamara at the age of 40.

However when this adenine is 105 years old as shown by johnson nepal in nature 2012. The presence of the a673t mutation also known as the icelandic mutation reduce the secretion of a beta 40 in our beta 42 peptide for the wild-type eppg and for appg containing the london mutation our experiments have shown that the presence of the a673t mutation reduce the secretion of a beta 40 and a beta 42 peptide by the epp genes not only for the wild-type gene but also for app genes containing several familial alzheimer's disease mutations as mentioned before the crispr cas9 base editing technology permit to modify the city into a timing we just have used the base editing technology to target the cytoplane in the antecedent strand of the alanine codon transforming that cytodine into a timing and just changing the alanine codon into a triangle the main problem with this approach is that although we want to modify the cytodine into the end descent strand of the adenine codon there are other acidity nucleotides nearby that are also affected by the base editing approach we have constructed 14 different base editing enzymes to be able to modify more specifically in the antisense cooldown of anatomy modifying the city and the antisense codon of led we have been able to introduce the h673t mutation in up to 17 of the avpg however other city also located in the antecedent strat were also modified by this approach a new fantastic technology called private has recently been developed by enceladus with permits in principle to replace any nucleotide by any the other editing technology use a cas9 decays fused with a reverse transcriptase it also requires a prime editing guide rna known as a pig remedy the peg rna is essentially prolonged single guide rna because as a single guide rna it contains a spacer sequence which react with 20 nucleotide in the target dna it also contains the constant scaffold of the single guide rna which is in red and then at its uh five prime end there is a prolongation with the primer bending side which is a sequence of 10 to 17 nucleotide reacting with the upper strand of dna this is followed by the reverse transcriptase template again which is 10 to 17 nucleotides in length and which will contain some modified nucleotide in red in this case indicating which nucleotide has to be modified by the reverse transcriptase a plasmid designed by anzalone at all is available at edgy to construct new peg rna this classmate contains a red fluorescent protein gene which may be removed by bsa1 cuts which produce a backbone of the protein and then the other components are the spacer primer binding side the reverse transcriptase template and the pig rna scalpel all of these sequence are single stranded oligonucleotide that may be purchased from idt these four parts are then assembled together to produce a new pegaron to use the prime editing technology we first have to identify photo spacer adjacent tab which is njj for the cast nine of streptococcus pyrogene this will permit to the cas9 to bind to the dna then the spacer sequence of the peg rna will bind to a 20 nucleotide sequence of dna in this case the lower strand and the formation of the complex between the peg rna class 9 decays and the dna will induce the nick at exactly 3 nucleotides from the pan and the upper strand of dna this will release the upper strand of dna to be able to interact with the primer binding side of the peg rna and then the reverse transcriptase template which contained few nucleotides to be related will be available for the reverse transcriptase to synthesize a new dna upper strand many duchenne muscular dystrophy patients have a stop codon dmdg since we did not have access to cells of patient containing such pointation we decided to introduce these stop codons using the prime editing technology so each case we had to identify an ngg identify the peg rna protospacer sequence and then modify the reverse transcriptase template so as to enter to modify a codon for an amino acid into a stockholder in this case we have changed to introduce the tga stuff we have successfully used that approach to introduce stun codon exam 9 20 35 42 55 and 61.

Have just designed various pig rna targeting dmd exon 35 as you can see we have varied the reverse transcriptase template in blue from 10 to 16 nucleotide and we have also varied primary binding site in green from 10 to 15 nucleotides and at the desired mutation site we have introduced a t to introduce that mutation our initial experiments were done in hgk 293 cells we tried to reproduce the mutation of mx1g and to target dmd exon 35.

So essentially these cells were transfected with plasmid coding for the cas9 djs fuse with the reverse transmitted days and a paid rna targeting either exon 35 of the dmd gene or the emh one gene the dna was extracted three days later and the targeted sequence was pcr amplified and sequenced using center method the sequence were analyzed using the edit r online program essentially we observe a 32 correction mutation of the air x1 gene as done by heads alone at all however for the exon 35 mutation we had a 2 background in the sequence of the control negative control and with the different peg rna we have mutation ranging from four to eight percent so this was not as great as for the e mx one g we have just tried different methods to try to increase the percentage of genome editing of exon 35.

The first method that we have tried is to repeat the treatment three times essentially the cells were transfected with plasmid at day 0 6 12. And dna was extracted three days and six days after each treatment we have amplified the pcr exon 35 at each of the extraction date and sequence it by sender and analyzer sequence as you can see the percentage of genome editing increased from day 3 to day 18 with the repeated treatment and this was the case for all three peg rna that were targeting we have then tested a second method to try to increase the percentage of mutation in exon 35.

It is to induce a second dig in the target gene this is the pe3 method essentially we have identified two pam sequence which permitted to kite rna to induce a second nick and either 57 nucleotides from the original big heavenly name or at 24 cleotide from the pagan herd when we induced a second leg at 24 nuclear time or at 57 nucleotides induced by the peg allergy there was a significant increase in the addition of the target gene for pig iron in the 35 4 and pig rna 256 but not for bigger and e35 you still have to understand why a third method to improve the percentage of mutation in the target gene is to mutate the pan used by the peg harmony we just designed pig rna that were not only able to introduce a stop codon mutation but we're also able to mutate simultaneously the pan and the use mutation of the pan used by the peg rna improve the percentage of mutation in the stop codon for two of the three peg rna that we have tested combining the two method that is inducing a second nick in the target g and mutating the pan used by the peg rna further increase the mutation of dmd exon 35 to 39 with all three peg that we have tested we are currently starting new project to correct mutation responsible for other heading steady disease we are working on cystic fibrosis due to mutation in the cftr chloride channel on congenital muscular dystrophy due to mutation in the rheanodine receptor and on ataxia 8 due to mutation in the nkx6 type 2 gene for each period steady disease due to a point mutation for example here with attacks at type 8 it is possible to correct in principle the mutated gene using the prime editing technology in this case here we can identify that the mutation is an adenosine change for timing and thus we can identify a pam njg for the sp cast iron which is close to the mutated nucleotide we can then design a plane rna that will introduce the desired mutation in this case we are introducing two mutations one to correct the mutation to reverse the timodine into an adenosine and the second midpoint mutation is to modify the path so that following the correction the cad9 enzyme can no longer bind to the dna crispr cas9 derived technologies may not be used to treat many individual diseases the main problem remains the inhibitor delivery of the editing agents thank you for your attention okay thank you professor jax uh i'll take the turn now to make a general comment for those who the who doesn't speak uh those who don't speak english okay thank you for your presentation.

Desdobramento em Medicina Baseada em Evidências: Correlação Fisiopatológica e Conduta Clínica

Sob os princípios da Medicina Baseada em Evidências (MBE) aplicados à Alergia e Imunologia Clínica, a interpretação de sinais e sintomas respiratórios, cutâneos, gastrointestinais ou sistêmicos exige a correlação rigorosa entre a história clínica detalhada (fenótipo) e os mecanismos imunológicos subjacentes (endótipo — mediado por IgE, por linfócitos T, por imunocomplexos ou por vias da imunidade inata). A investigação diagnóstica direcionada — utilizando testes cutâneos padronizados, dosagens laboratoriais específicas e provas funcionais — permite estabelecer o diagnóstico diferencial preciso, evitar a automedicação e instituir um plano terapêutico individualizado voltado ao controle sustentado da inflamação e à preservação da qualidade de vida do paciente.

Abordagem Integrada em Alergia e Imunologia Clínica: Diagnóstico e Prevenção

Compreendendo a Hipersensibilidade Imunológica e Seus Fatores Modificáveis

As manifestações alérgicas e imunomediadas resultam da interação dinâmica entre herança genética, barreira epitelial (respiratória, cutânea e digestiva) e exposição contínua a fatores ambientais do cotidiano. Diferentemente de quadros infecciosos autolimitados, os processos de hipersensibilidade tendem à recorrência quando apenas os sintomas superficiais são medicados de forma paliativa, tornando indispensável compreender o mecanismo imunológico subjacente — seja ele mediado por anticorpos IgE, células T ou vias inflamatórias mistas.

Investigação Especializada, Testes Alérgicos e Imunoterapia

O caminho mais seguro para recuperar o bem-estar inicia-se pela anamnese detalhada associada ao teste de alergia adequado a cada suspeita clínica. Com o diagnóstico etiológico estabelecido, estratégias modernas como a vacina para alergia e o tratamento de rinite das condições respiratórias ou cutâneas correlatas permitem controlar a inflamação de base, reduzir o uso repetitivo de medicamentos sintomáticos e promover qualidade de vida duradoura.

Importância do Diagnóstico Diferencial e da Anamnese Detalhada

Na especialidade de Alergia e Imunologia, dois pacientes com sintomas aparentemente semelhantes envolvendo Parte 3 podem apresentar mecanismos fisiopatológicos completamente distintos. Por essa razão, a história clínica minuciosa — investigando cronologia das crises, ambiente domiciliar e profissional, histórico familiar de atopia, uso de fármacos concomitantes e hábitos alimentares — funciona como bússola indispensável para interpretar corretamente exames laboratoriais (como dosagem de IgE total e específica) e provas funcionais. Um resultado laboratorial isolado jamais substitui o julgamento clínico presencial ou por telemedicina especializada, prevenindo diagnósticos equivocados e tratamentos desnecessários.

Prevenção Ambiental, Adesão Terapêutica e Monitoramento Contínuo

O sucesso a longo prazo no manejo das condições alérgicas e imunológicas depende diretamente da aliança entre tratamento médico direcionado e medidas educativas cotidianas. Manter ambientes bem ventilados e livres de acúmulo de poeira doméstica, preservar a hidratação fisiológica das mucosas e da barreira cutânea, respeitar rigorosamente os horários e a técnica de uso das medicações preventivas prescritas e realizar revisões periódicas permitem estabilizar a inflamação subclínica antes que ela evolua para exacerbações agudas.

Critérios de Segurança Clínica e Sinais de Alerta em Alergologia

Reconhecer precocemente os sinais de alerta que exigem reavaliação médica prioritária faz parte da educação em saúde de todo paciente e familiar. Sintomas como dispneia progressiva (dificuldade para respirar ou falar frases completas), chiado torácico refratário, edema súbito de lábios, língua ou glote, tontura por queda pressórica, febre persistente associada a lesões de pele ou acometimento de mucosas demandam atendimento imediato de urgência, seguido de investigação alergológica estruturada para prevenir recorrências futuras.

Impacto na Qualidade de Vida, Sono e Bem-Estar Sistêmico

Além das manifestações físicas diretas, quadros não controlados ligados a Parte 3 interferem profundamente na arquitetura do sono reparador, na capacidade de concentração escolar e profissional e no equilíbrio emocional. O estresse crônico, por meio de mediadores neuroendócrinos e alarminas epiteliais, retroalimenta a resposta inflamatória imunitária. Dessa forma, instituir uma abordagem integral de reabilitação clínica e ambiental restaura a autonomia diária e o bem-estar global do indivíduo.

Perguntas Complementares Frequentes na Prática Clínica

Reações Imunológicas a Picadas de Insetos e Prevenção de Crises: As reações desencadeadas por artrópodes dividem-se clinicamente entre hipersensibilidade à saliva de insetos hematófagos (como pernilongos, borrachudos e pulgas, causadores do prurigo estrófulo na infância) e alergia sistêmica à peçonha de himenópteros (abelhas, vespas e formigas-lava-pés). Enquanto os antígenos salivares costumam gerar pápulas pruriginosas persistentes e risco de infecção bacteriana secundária pelo ato de coçar, o veneno injetado por ferrão pode deflagrar degranulação maciça de mastócitos mediada por anticorpos IgE, exigindo avaliação da triptase sérica basal e plano de ação de emergência.

Mecanismos da Alergia Alimentar, Diagnóstico Preciso e Manejo Nutricional: O sistema imunológico associado à mucosa gastrointestinal atua normalmente mantendo tolerância oral diante de milhares de proteínas da dieta. Quando há quebra dessa barreira imunitária — influenciada por predisposição genética, disbiose da microbiota intestinal ou imaturidade da IgA secretora na infância —, frações proteicas intactas sensibilizam mastócitos e basófilos. Por isso, um exame de sangue isoladamente positivo reflete apenas sensibilização biológica, devendo sempre ser correlacionado à história clínica e, quando necessário, ao teste de provocação oral supervisionado em ambiente médico.

Imunologia da Mucosa Digestiva: Eixo Intestino-Vias Aéreas e Diagnóstico Diferencial: As afecções imunomediadas do trato gastrointestinal — incluindo a esofagite eosinofílica, a doença celíaca e a sensibilidade não celíaca ao glúten — compartilham alterações na integridade das junções firmes do epitélio digestivo. Na esofagite eosinofílica, frequentemente associada a rinite, asma e dermatite atópica, a infiltração de eosinófilos na parede do esôfago provoca disfagia, impactação alimentar e sintomas que simulam refluxo gastroesofágico refratário aos inibidores de bomba de prótons.

Saúde da Barreira Cutânea, Inflamação Imunológica e Controle do Prurido: Nas dermatites eczematosas e quadros de prurido crônico, diferenciar a atopia constitucional da dermatite alérgica de contato (uma reação de hipersensibilidade tardia do tipo IV mediada por linfócitos T sensibilizados a metais, conservantes cosméticos, fragrâncias, resinas ou borracha) é indispensável. A exposição repetida ao agente sensibilizante mantém a inflamação cutânea ativa mesmo sob uso de pomadas anti-inflamatórias, exigindo identificação exata do fator desencadeante.

Imunologia das Doenças Autoimunes Cutâneas e Cuidado Integrado: Diferentemente das dermatoses puramente mediadas por IgE, a psoríase e as condições correlatas de autoagressão envolvem ativação de células dendríticas plasmocitoides, produção de interferon-gama e estímulo sustentado do eixo interleucina-23 / interleucina-17 (IL-23/IL-17) e TNF-alfa. Essa sinalização acelera em várias vezes o ciclo de renovação dos queratinócitos da epiderme, originando placas eritemato-descamativas bem delimitadas e, em parte dos pacientes, inflamação articular associada. Infecções, traumas mecânicos ou químicos na pele (fenômeno de Koebner), clima seco de inverno e estresse emocional funcionam como importantes amplificadores da resposta inflamatória.

Fisiopatologia das Urticárias e do Angioedema: Diagnóstico e Controle Seguro: Quando ocorre angioedema isolado recorrente sem placas pruriginosas ao redor, o raciocínio imunológico deve distinguir os quadros histaminérgicos daqueles mediados por bradicinina — incluindo o angioedema hereditário ou adquirido por deficiência funcional ou quantitativa do inibidor de C1-esterase (C1-INH) e as reações induzidas por anti-hipertensivos inibidores da enzima conversora de angiotensina (IECA). Essa diferenciação é vital, pois o angioedema bradicininérgico não responde a anti-histamínicos ou corticoides convencionais.

Controle da Inflamação Brônquica, Função Pulmonar e Prevenção do Remodelamento: A asma brônquica e a bronquite alérgica constituem doenças inflamatórias crônicas das vias aéreas inferiores, marcadas por hiper-reatividade brônquica, edema de mucosa, hipersecreção de muco e obstrução variável ao fluxo expiratório. Quando a inflamação eosinofílica ou alérgica (tipo 2) permanece sem controle adequado ao longo dos anos — ou quando o paciente utiliza apenas broncodilatadores de alívio rápido sem tratar a base inflamatória —, ocorre espessamento da membrana basal e hipertrofia da musculatura lisa brônquica, processo conhecido como remodelamento brônquico.

Via Aérea Unificada: Manejo da Rinite, Rinossinusite e Hipersensibilidade Nasal: Sob o conceito médico de 'via aérea única', a inflamação não controlada das cavidades nasais e dos seios paranasais repercute diretamente nos ouvidos (disfunção tubária), nos olhos (rinoconjuntivite), na qualidade do sono e nos brônquios. Em subgrupos específicos, como na Doença Respiratória Exacerbada por Aspirina (DREA), soma-se a polipose nasal recidivante, a asma e a intolerância aos anti-inflamatórios inibidores da COX-1, exigindo diagnóstico especializado para restaurar o olfato e a patência respiratória.

Segurança Farmacológica, Reações Adversas a Medicamentos e Uso Racional de Corticoides: As reações de hipersensibilidade a fármacos abrangem mecanismos imunológicos e farmacológicos distintos: desde reações imediatas (até uma a seis horas após a dose, manifestadas por urticária, angioedema labial ou palpebral, broncoespasmo e anafilaxia, comuns com analgésicos inibidores da COX-1 e betalactâmicos) até farmacodermias tardias mediadas por células T, como exantemas maculopapulares e a síndrome DRESS — condição sistêmica que surge semanas após o início de anticonvulsivantes, sulfonas ou alopurinol, cursando com febre, linfonodomegalia, eosinofilia e necessidade de suspensão imediata sob supervisão hospitalar.

Imunização Segura, Resposta Imunológica e Proteção do Paciente Alérgico: Na avaliação da competência do sistema imune — seja em infecções de repetição, alterações laboratoriais de imunoglobulinas (IgG, IgA, IgM, IgE) ou em pessoas vivendo com HIV sob acompanhamento da contagem de linfócitos T CD4 —, distingue-se criteriosamente o uso de vacinas inativadas/subunidades (amplamente seguras mesmo em imunossuprimidos) das vacinas de agentes vivos atenuados, cuja indicação depende do grau de preservação da imunidade celular. Ademais, pacientes em uso de imunoterapia antialérgica podem manter seu calendário vacinal respeitando apenas pequenos intervalos operacionais orientados pela equipe médica.

Medicina de Precisão: Como Funcionam os Anticorpos Monoclonais e Imunobiológicos: Os anticorpos monoclonais (imunobiológicos) revolucionaram o manejo das doenças alérgicas e imunomediadas graves que não alcançam controle pleno com terapias convencionais. Diferentemente dos corticoides sistêmicos e imunossupressores clássicos — que deprimem amplamente a imunidade e geram toxicidade metabólica —, os imunobiológicos atuam como chaves de alta precisão molecular, neutralizando alvos específicos da cascata inflamatória: como a IgE livre circulante (omalizumabe), o receptor de IL-4 e IL-13 (dupilumabe), a via eosinofílica da IL-5 (mepolizumabe e benralizumabe) ou a alarmina epitelial TSLP (tezepelumabe).

Conclusão e Síntese das Orientações

Em síntese, a abordagem adequada de Parte 3 exige compreensão clara dos mecanismos imunológicos envolvidos, diagnóstico etiológico preciso e acompanhamento médico individualizado. Evitar a automedicação e investigar a causa raiz dos sintomas — contando com recursos especializados e orientações seguras como o cuidado especializado em alergia e imunologia quando clinicamente pertinente — é o caminho mais seguro para controlar crises, prevenir complicações e assegurar qualidade de vida sustentável.

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