Perspektive u razvoju profilaktičkih vakcina

Vaccines are considered to be one of the greatest public health achievements of the last century. As a result of widespread vaccine use, the smallpox virus has been completely eradicated and the incidence of other diseases such as polio, measles, tetanus and diphtheria has been drastically reduced. Current licensed vaccines, predominantly composed of either live attenuated or killed pathogens, pathogen subunits, owe their success to their ability to elicit neutralizing antibodies against pathogens. On the other side, cell-mediated immunity, which plays a central role in elimination of intracellular pathogens (which in most cases leads to chronic infections) is much more difficult to obtain using current vaccines. Currently, numerous vector and nucleic acid (DNA and mRNA)-based prophylactic vaccines, capable of inducing substantial vaccine-specific T cell responses, are investigated in preclinical and clinical studies, with promising results. This review focuses the background of vector and nucleic acid-based vaccines, their strengths and weaknesses and safety issues.Vakcine se smatraju jednim od najvećih javnozdravstvenih dostignuća prošlog veka. Zahvaljujući vakcinaciji u svetu su potpuno iskorenjene velike boginje, a incidencija drugih infektivnih bolesti, kao što su dečija paraliza, male boginje, tetanus i difterija, drastično je smanjena. Današnje licencirane vakcine, koje pretežno sadrže žive atenuisane ili mrtve patogene ili njihove delove, su uspešne zahvaljujući tome što stimulišu produkciju neutrališućih antitela. Sa druge strane, ove vakcine mnogo teže indukuju ćelijski-posredovanu imunost, koja je važna za eliminaciju intraćelijskih patogena (koji često dovode do hroničnih infekcija). Trenutno se u pretkliničkim i kliničkim studijama ispituju brojne profilaktičke vakcine zasnovane na vektorima i nukleinskim kiselinama (DNK i iRNK), sposobne da indukuju snažan odgovor T-ćelija na vakcinalni antigen, sa obećavajućim rezultatima. U ovom radu su date osnovne informacije o vakcinama sa vektorima i nukleinskim kiselinama, opisani su mehanizmi kojima one pokreću imunski odgovor, njihove dobre i loše strane, kao i problemi vezani za njihovu bezbednu primenu

Farmakogenetika i farmakogenomika - uticaj jednonukleotidnih polimorfizama na odgovor na lekove

Individual variation in response to drugs is an important clinical problem, which ranges from failure to respond to the drug, over adverse reactions to drugs, to interactions among drugs being administered concurrently. Numerous findings indicate that the differences in the patients response on the same drug are caused by genetic variations. This is the subject of pharmacogenetics. Although pharmacogenetics generally equated with the concept of pharmacogenomics, pharmacogenetics is primarily related to variations in a single gene that influence the on drug response, while pharmacogenomics is a broader term, which studies how all of the genes (the genome) can influence responses to drugs. In focus of this paper will be individual variation in response to drugs arising from single nucleotide polymorphisms in genes encoding the drug target proteins, enzymes that metabolize drugs, drug transporters, and polymorphisms of genes responsible for toxicity and hypersensitivity to drugs. Determination of pharmacogenetic profile of patients could point out patients who are at increased risk of adverse drug effects (for which drug should be applied at lower doses or other drugs can be used) and those in which are likely to achieve the desired therapeutic effect, and so to enable individualization of therapy.Individualne varijacije u odgovoru na lekove važan su klinički problem i mogu dovesti do potpunog odsustva reakcije na lek i pojave neželjenih reakcija na lekove. Brojni nalazi ukazuju da su razlike u odgovoru bolesnika na isti lek uslovljene genetskim varijacijama, i predmet su istraživanja farmakogenetike. Iako se farmakogenetika uglavnom izjednačuje sa pojmom farmakogenomika, farmakogenetika se uglavnom odnosi na varijacije u jednom genu koje utiču na odgovor na lek, dok je farmakogenomika šira oblast, koja ispituje kako svi geni u genomu povezani sa metabolizmom određenog leka mogu uticati na odgovor na dati lek. U ovom radu prevashodno će biti opisane individualne varijacije u odgovoru na lekove koje nastaju usled jednonukletidnih polimorfizama u genima koji kodiraju ciljne proteine lekova, enzime koji metabolišu lekove, transportere lekova, kao i polimorfizmi gena koji su odgovorni za toksičnost i preosetljivost na lekove. Određivanje farmakogenetskog profila bolesnika moglo bi da ukaže na bolesnike koji su u povećanom riziku od pojave neželjenih efekata lekova (kod kojih bi trebalo da se primene niže doze ili drugi lekovi) i na one kod kojih će se najverovatnije postići željeni terapijski efekat, odnosno da omogući individualizaciju terapije

Imunološke i molekularne tehnike u laboratorijskoj dijagnostici virusnih infekcija

Diagnostic virology is presently integrated into routine medical practice. The expanded role of diagnostic virology has several explanations. First, the human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) epidemic and the success of organ transplantation have greatly increased the pool of patients at risk for serious opportunistic viral infections. Second, an increasing number of antiviral agents are available, and their use often depends on establishing a laboratory-based diagnosis. Third, technologic developments such as monoclonal antibodies and the polymerase chain reaction (PCR) have made rapid viral diagnosis a reality. An important characteristic of modern diagnostic virology is the use of multiple methods for detecting viral infections. Viral culture, detection of viral antigens and nucleic acids, and detection of viral antibodies are all used to diagnose current viral infection. Many of molecular techniques for detecting of viral genomes are in use in routine diagnostic laboratory to aid viral diagnosis. PCR has been used to detect numerous viruses and is increasingly used to assess prognosis, monitor response to treatment, assess progession of infection, and assess of viral resistance to antiviral drugs. The sensitivity and specificity of PCR and other Nucleic Acid Amplification Techniques (NAATs) have made NAATs the diagnostic assay of choice in routine diagnostic laboratories to replace other less sensitive methods of viral detection.Laboratorijska dijagnostika virusnih infekcija danas je integralni deo medicinske prakse. Sve veća uloga virusološke dijagnostike objašnjava se: i) epidemijom sindroma stečene imunodeficijencije (AIDS), kao i napretkom u transplantaciji organa, što je dovelo do značajnog porasta broja pacijenata koji su podložni razvoju oportunističkih virusnih infekcija, ii) sve većim brojem dostupnih antivirusnih lekova, a njihova primena zavisi od laboratorijski potvrđene dijagnoze, i iii) tehnološkim napretkom i razvojem novih tehnika, kao što su produkcija monoklonskih antitela i lančana reakcija polimeraze (PCR), koje su omogućile brzu dijagnostiku virusnih infekcija. Važna karakteristika moderne dijagnostičke virusologije je korišćenje različitih metoda u detekciji virusnih infekcija. Kultura virusa, detekcija virusnih antigena i nukleinskih kiselina, kao i detekcija virusnih antitela, danas se primenjuju za dijagnozu virusnih infekcija. Mnoge molekularne tehnike za detekciju virusnih genoma primenjuju se u rutinskim dijagnostičkim laboratorijama za postavljanje dijagnoze virusnih infekcija. PCR se koristi za detekciju brojnih virusa, a sve više i za određivanje prognoze bolesti, praćenje uspešnosti primenjene terapije, procenu progresije infekcije i određivanje rezistencije virusa na anti-virusne lekove. Osetljivost i specifičnost PCR i drugih tehnika amplifikacije nukleinskih kiselina (NAATs), učinile su da ove tehnike postanu metode izbora u rutinskim dijagnostičkim laboratorijama i da potisnu ostale manje osetljive metode za detekciju virusa

Miokarditis - značaj eksperimentalnih modela i autoimunskih mehanizama u etiopatogenezi bolesti

Acute myocarditis is a serious disease and a major cause of dilated cardiomyopathy. Patients with myocarditis may present severe arrhytmia , rapidly progressive heart failure or shock. In humans, about half the cases of myocarditis are preceded by an acute viral infection. Autoimmunity is important in myocarditis: in particular, a reaction to cardiac myosin following viral infection may contribute to development of myocarditis. Experimental autoimmune myocarditis (EAM) is used as an animal model of human giant cell myocarditis. EAM is characterized by severe myocardial damage that includes the infiltration of mononuclear cells into the myocardium and the appearance of multinucleated giant cells. Treatment of acute myocarditis in humans remains a major clinical problem. Although myocardial inflammation and autoimmune process are essential for the progression of the disease, most results do not support routine treatment of myocarditis with immunosuppressive drugs.Akutni miokarditis je klinički ozbiljno oboljenje i glavni uzrok dilatacione kardiomiopatije. Kod pacijenata sa miokarditisom može se u kratkom vremenskom periodu razviti aritmija, progresivna srčana insuficijencija i šok. Polovina svih slučajeva miokarditisa kod čoveka javlja se nakon akutne virusne infekcije miokarda. Pored direktnog oštećenja mišićnih ćelija srca virusom i autoimunski odgovor organizma na miozin, koji se oslobađa iz kardiomiocita tokom virusne infekcije, ima važnu ulogu u patogenezi miokarditisa. Eksperimentalni autoimunski miokarditis (EAM) predstavlja model koji odgovara miokarditisu džinovskih ćelija kod ljudi. EAM karakteriše difuzna mononuklearna ćelijska infiltracija, intersticijski edem i masovna nekroza kardiomiocita. U pojedinim infiltratima se zapažaju karakteristične džinovske višejedarne ćelije. Lečenje akutnog miokarditisa i dalje predstavlja značajan klinički problem. Iako su inflamacija i autoimunski odgovor ključni faktori u razvoju i progresiji bolesti, većina kliničkih studija nije potvrdila opravdanost rutinske primene imunosupresivnih lekova u terapiji miokarditisa

Razvoj monoklonskih antitela za terapijsku primenu - od mišjih do humanih

Therapeutical monoclonal antibodies (mAbs) represent one of the fastest growing area of the pharmaceutical industry. High target specificity and specific molecular structure are features that make antibodies attractive drug candidates for therapeutical use. Besides high specificity, other characteristics including immunogenicity, affinity, effector functions, half-life as well as easy of production, stability and cost must be considered when an antibody is designed as drug. In this paper the structure and function of antibody, new technologies for the generation of partly and fully human mAbs and their fragments and methods for enhancing their affinity, effector functions and pharmacokinetics will be describe.Monoklonska antitela (mAt) i njihovi derivati su najveća grupa proteina koji se koriste u terapiji i predstavljaju segment farmaceutske industrije koji se najbrže razvija. Osobine antitela koje ih čine izuzetno interesantnim za primenu u terapiji su prvenstveno njihova visoka specifičnost za ciljni molekul a zatim i njihova karakteristična građa. Pored visoke specifičnosti, i druge osobine antitela kao što su imunogenost, afinitet, stabilnost, efektorske funkcije, poluživot, penetracija i distribucija u tkiva, moraju se uzeti u obzir kada se ove molekule koriste kao lekovi. Sa stanovišta proizvodnje, što lakše dobijanje, stabilnost i manja cena su najvažniji zadaci koje farmaceutska industrija treba da ostvari. U ovom radu biće opisana građa i funkcija antitela, savremene metode za dobijanje delimično i kompletno humanih mAt i njihovih derivata, kao i načini za poboljšanje njihovog afiniteta, efektorske funkcije i farmakokinetike

A monoclonal antibody to the rat Crry/p65 antigen, a complement regulatory membrane protein, stimulates adhesion and proliferation of thymocytes

A murine monoclonal antibody (mAb), 3F10, was produced by fusion of spleen cells obtained from mice immunized with a rat cortical thymic epithelial cell line (R-TNC.1) stimulated with interferon-gamma and P3X myeloma cells. 3F10 recognized an antigen expressed both on thymocytes and non-lymphoid cells in the thymus. Flow cytometry showed that 3F10 stained more than 98% thymocytes and 90% R-TNC.1 cells. Immunoprecipitation and Western blot studies demonstrated that 3F10 reacted with molecules of 55000 and 65000 MW from both thymocyte and R-TNC.1 cell lysates. 3F10 recognized the same antigen on Chinese hamster ovary cells transfected with rat Crry as did 5I2 mAb, confirming the specificity of 3F10 mAb for the rat homologue of mouse Crry/p65, a membrane-bound complement regulatory protein. 3F10 mAb induced homotypic aggregation of thymocytes and exhibited an additive effect on the aggregation evoked by phorbol myristate acetate. The aggregation was dependent on active cell metabolism, intact cytoskeleton, divalent cations and activation of protein phosphatases 1 and 2A (as assessed by use of okadaic acid). In contrast, H-7, HA1004 and genistein partially inhibited, whereas staurosporine potentiated the aggregation of thymocytes triggered by 3F10. 3F10 mAb also stimulated binding of thymocytes to the R-TNC.1 line. Both homotypic and heterotypic adhesive interactions are mediated by leucocyte function-associated antigen-1 (LFA-1). In addition, 3F10 stimulated proliferation of thymocytes induced by suboptimal concentrations of concanavalin A. These data suggest that rat Crry/p65 might be involved in the regulation of both cell adhesion and activation of thymocytes. This is a novel, non-complement-dependent function of Crry/p65

Neželjeni efekti bioloških lekova - reakcije preosetljivosti

Clinical use of various biological agents (protein-based products derived from biological sources, such as bacteria, fungi or mammalian cells) have greatly improved the treatment of a wide variety of diseases in humans. Nevertheless, a variety of their adverse reactions have been observed. These adverse effects have been clasified in five types (α, β, γ, δ and ε). Type β adverse effect involves hypersensitivity reactions linked to the immunogenicity of biological agents. It is worth emphasizing that biological agents irrespective whether they contain xenoantigens may lead to development of anti-drug antibodies. Several drug-, patients-, diseaseand treatment-related factors contribute to immunogenicity of biological agents. Anti-drug antibodies can influence either the efficacy of biological agent or induce hypersensitivity reactions. One of them is immediate infusion reaction, which occurs during or within one hour after biological agent infusion. This reaction may cause mild to severe clinical manifestations. Involvement of IgE- and non-IgE isotypes in hypersentitivity reactions are confirmed by novel methods for detecting anti-drug antibodies. This manuscript summarizes the current knowledge on factors influencing immunogenicity to biological agents, and points to the mechanisms underlying the drug-induced hypersensitivity reactions.Više od dve decenije biološki lekovi (proteini dobijeni iz bioloških izvora, kao što su bakterije, gljive ili ćelije sisara) koriste se u terapiji različitih autoimunskih/ inflamatornih bolesti i tumora kod ljudi. Primena ovih lekova praćena je pojavom novih neželjenih efekata koji se mogu svrstati u pet različitih grupa koje se obeležavaju slovima grčkog alfabeta (od α do ε). Reakcije preosetljivosti koje se vezuju za imunogenost bioloških lekova su klasifikovane kao reakcije iz grupe tip β. U praksi, svi proteini koji se koriste kao biološki lekovi, pokreću imunski odgovor, a kao rezultat nastaju anti-lek antitela. Brojni faktori koji su vezani za sam lek, pacijenta ili način davanja leka mogu da utiču na imunogenost nekog biološkog leka. Anti-lek antitela mogu da se vežu za lek i smanje njegovu efikasnost ili da izazovu reakcije preosetljivosti (uglavnom tip I, ređe tip III). Infuzijska reakcija, koja se javlja već u toku davanja infuzije leka ili u okviru jednog sata od njenog završetka, jedna je od najčešćih reakcija preosetljivosti na biološke lekove. U ovom radu opisani su faktori koji mogu da utiču na imunogenost bioloških lekova i patogenetski mehanizmi reakcija preosetljivosti na biološke lekove

Normalna ljudska mikrobiota i disbioza - implikacije po zdravlje i bolest

The normal human microbiota, formerly called the "microbial flora," consists of bacteria, fungi, viruses, and parasites that colonise the skin and mucous membranes of the respiratory, gastrointestinal, and genitourinary tracts. The number and diversity of microorganisms varies between different body niches and is greatest in the intestinal tract. The microbiota contributes to the homeostasis of the human organism by preventing colonisation by pathogenic microorganisms, participating in digestive processes and metabolism, and regulating immune functions. Various environmental and genetic factors can lead to an imbalance in the human microbiota, called dysbiosis, which can affect human health. Dysbiosis is usually the result of decreased microbial diversity and a lower number of saprophytic microorganisms, followed by an overgrowth of opportunistic species. The most common diseases directly related to intestinal dysbiosis are antibiotic-associated diarrhoea and pseudomembranous colitis, both of which are associated with the excessive growth of harmful bacteria and Clostridioides difficile following broad-spectrum antibiotic therapy. Dysbiosis is associated with various health conditions or diseases such as acne, psoriasis, eczema, chronic obstructive pulmonary disease, inflammatory bowel disease, obesity, metabolic syndrome, type 2 diabetes, autoimmune diseases and allergies, neurological diseases such as Parkinson's disease, Alzheimer's disease, epilepsy and stroke, depression, anxiety, infertility, preterm birth, and malignancies.Normalna ljudska mikrobiota, koja se ranije nazivala „mikroflora“, sastoji se od bakterija, gljivica, virusa i parazita koji kolonizuju kožu i sluzokožu respiratornog, gastrointestinalnog i genitourinarnog trakta. Broj i raznovrsnost mikroorganizama variraju između različitih telesnih niša i najveći su u crevnom traktu. Mikrobiota doprinosi homeostazi ljudskog organizma tako što sprečava kolonizaciju patogenim mikroorganizmima, učestvuje u procesima varenja i metabolizma i reguliše imunološke funkcije. Disbioza je stanje u kome dolazi do neravnoteže sastava mikrobiote usled uticaja različitih egzogenih ili endogenih faktora, što može uticati na ljudsko zdravlje. Ona je najčešće rezultat smanjene raznovrsnosti mikroorganizama i manjeg broja saprofitnih bakterija, što je praćeno prekomernim rastom potencijalno štetnih vrsta. Najčešće bolesti koje su direktno povezane sa crevnom disbiozom su dijareja povezana sa primenom antibiotika i pseudomembranozni kolitis, a obe nastaju kao posledica prekomernog rasta štetnih bakterija i Clostridioides difficile nakon terapije antibioticima širokog spektra. Disbioza je povezana sa različitim zdravstvenim stanjima ili bolestima kao što su akne, psorijaza, ekcem, hronična opstruktivna bolest pluća, inflamatorna bolest creva, gojaznost, metabolički sindrom, dijabetes tipa 2, autoimunske bolesti i alergije, neurološke bolesti kao što su Parkinsonova bolest, Alchajmerova demencija, epilepsija i moždani udar, depresija, anksioznost, neplodnost, prevremeni porođaj i maligni tumori

Sex differences in Tfh cell help to B cells contribute to sexual dimorphism in severity of rat collagen-induced arthritis

The study examined germinal centre (GC) reaction in lymph nodes draining inflamed joints and adjacent tissues (dLNs) in male and female Dark Agouti rat collagen type II (CII)-induced arthritis (CIA) model of rheumatoid arthritis. Female rats exhibiting the greater susceptibility to CIA mounted stronger serum CII-specific IgG response than their male counterparts. This correlated with the higher frequency of GC B cells in female compared with male dLNs. Consistently, the frequency of activated/proliferating Ki-67+ cells among dLN B cells was higher in females than in males. This correlated with the shift in dLN T follicular regulatory (Tfr)/T follicular helper (Tfh) cell ratio towards Tfh cells in females, and greater densities of CD40L and CD40 on their dLN T and B cells, respectively. The higher Tfh cell frequency in females was consistent with the greater dLN expression of mRNA for IL-21/27, the key cytokines involved in Tfh cell generation and their help to B cells. Additionally, in CII-stimulated female rat dLN cell cultures IFN-gamma /IL-4 production ratio was shifted towards IFN-gamma. Consistently, the serum IgG2a(b)/IgG1 CII-specific antibody ratio was shifted towards an IgG2a(b) response in females. Thus, targeting T-/B-cell interactions should be considered in putative further sex-based translational pharmacology research

Neonatal castration affects intrathymic kinetics of T-cell differentiation and the spleen T-cell level

To test putative interdependence in the ontogenesis of the hypothalamic-pituitary-gonadal and thymic-lymphatic axes, thymocyte differentiation and maturation was examined in neonatally castrated (Cx) adult rats. In the hypercellular thymi of Cx rats, the proportion of the least mature CD4(-)CD8(-)TCR alpha beta(-) triple negative (TN) thymocytes was reduced, while the proportions of all downstream double positive (DP) subsets (TCR alpha beta(-), TCR alpha beta(low) and TCR alpha beta(high)) were increased when compared with neonatally sham-castrated (Sx) adult rats. This suggested an accelerated thymocyte transition from the TN to DP TCR alpha beta(low) developmental stage accompanied by an increased positive/reduced negative thymocyte selection. The increased thymocyte surface density of Thy-1, which is implicated in thymocyte hyposensitivity to negative selection, in Cx rats further supports the previous assumption. The finding that the proportions of both single positive (SP) TCR alpha beta(high) thymocyte subsets were reduced, while their numbers were increased (CD4(+)CD8(-)) or unaltered (CD4(-)CD8(+)), coupled with results demonstrating an increased level of CD4(-)CD8(+) cells without changes in that of CD4(+) 8(-) cells in the spleen indicate: (i) accelerated differentiation and maturation of the positively selected DP TCR alpha beta(high) thymocytes towards CD4(-)8(+) TCR alpha beta(high) cells followed by increased emigration of the mature cells and (ii) decelerated hi h differentiation and maturation towards CD4(+)8(-) TCR alpha beta(high) cells in Cx rats. Furthermore, the unaltered proportion of intrathymically developing CD4(+)CD25(+)Foxp3(+) regulatory cells in Cx rats, in light of putative hyposensitivity of thymocytes to negative selection suggesting reduced elimination of autoreactive cells, may provide a firm basis for understanding the reasons behind increased susceptibility of Cx rats to autoimmune disease induction