Natural killer cell cytotoxic activity monitoring in selected prostate cancer patiens during the course of experimental immunotherapy

Naravne celice ubijalke (NK) so efektorske celice prirojenega imunskega sistema. Njihova ključna naloga je prepoznavanje in uničevanje rakavo spremenjenih ter z virusi in glivicami okuženih celic. Poleg tega so zgodnji vir provnetnih citokinov, zlasti interferona ? (IFN-?) in kemokinov, ki aktivirajo in privabljajo tudi druge celice imunskega sistema. V periferni krvi sta prisotni dve večji subpopulaciji celic NK, in sicer izrazito citotoksične CD56PdimP, ki so v večini ter regulatorne CD56PbrightP, ki jih je malo in so pomemben vir IFN-?. Aktivnost celic NK (NKA) lahko določamo tako z merjenjem sproščanja IFN-? in vitro kot s testom njihove neposredne citotoksičnosti zoper modelne tarčne celice. NKA je pri zdravih posameznikih skozi daljše časovno obdobje dokaj stabilna, prav tako pa se pri njih tudi razmerja med subpopulacijama celic NK ne spreminjajo bistveno. V literaturi večinoma poročajo o zmanjšanju vrednosti NKA z napredovanjem raka prostate, ob tem pa obstajajo tudi podatki o zmanjšanju deleža CD16P-PCD56Pbright Pcelic NK v periferni krvi. V okviru magistrske naloge smo določali NKA v vzorcih mononuklearnih celic iz periferne krvi, odvzetih šestim izbranim bolnikom z rakom prostate v različnih časovnih obdobjih, in sicer pred, med in po kliničnem preskušanju protitumorske imunske celične terapije. Odstotke citotoksičnosti celic NK v omenjenih vzorcih smo določali v pogojih in vitro, na osnovi rezultatov testa za ugotavljanje obsega neposredne celične citotoksičnosti, ki temelji na količini sproščenega znotrajceličnega encima laktatne dehidrogenaze (LDH) iz uničenih tarčnih celic K562. Za izvajanje testov smo na podlagi predhodnih poskusov izbrali mikrotitrske plošče s 96 vdolbinicami v obliki črke V. Pri dveh bolnikih smo za primerjavo vzporedno izvedli tudi test citotoksičnosti s pretočno citometrijo, pri katerem smo tarčne celice K562 označevali s fluorescenčnima barviloma CFSE in 7-AAD. Rezultati naših poskusov so v večini primerov pokazali precejšnja nihanja NKA tekom klinične študije, glede na kontrolne vrednosti odstotka citotoksičnosti, določene v vzorcih, odvzetih pred začetkom eksperimentalne imunske celične terapije, kar je najverjetneje posledica omenjenega zdravljenja. Izbrani bolniki so bili predstavniki treh skupin, ki so jih izvajalci klinične študije oblikovali glede na njihovo odzivnost na preskušano terapijo, ovrednoteno z definiranimi kliničnimi parametri. Povezave med klinično oceno njihove odzivnosti in NKA z našim omejenim številom poskusov nismo uspeli dokazati, vendar pa so se pokazali določeni trendi, ki nakazujejo na to, da bi nam tak dokaz uspel, če bi imeli na razpolago dovolj velik vzorec preiskovancev.Natural killer cells (NK) are effector cells of innate immune system. Their key role is to recognise and destroy cancerous, virus infected and fungus infected cells in humans. They are also an early source of proinflammatory cytokines, especially interferon ? (IFN-?) and chemokines that activate and attract other immune cells. There are two main subpopulations of NK cells in peripheral blood, distinctly cytotoxic subpopulation of CD56Pdim PNK cells and mostly regulatory CD56PbrightP NK cells that are an important source of early IFN-?. NK cell activity (NKA) can be determined by in vitro measuring of released IFN-? as well as with a direct cytotoxicity assay using model target cells. NKA in healthy individuals is relatively stable through longer periods of time and the ratio between NK subpopulations remains mostly unchanged. In clinical studies it is reported that in patients with prostate cancer, NKA decreases with advancing cancer stage. These studies are accompanied with reports of diminishing subpopulation of CD16P-PCD56Pbright Pin peripheral blood, which promotes a stronger immune response against cancer cells. In our work we measured cytotoxic NKA in samples of cells acquired from PBMC of six patients with prostate cancer in different time points during a clinical trial of an anticancer immune therapy. The percentages of cytotoxicity were measured in in vitro conditions on the basis of results from a direct cytotoxicity assay based on the amount of released intracellular enzyme lactate dehydrogenase (LDH) from killed target cells K562. We used a letter V shaped 96 well microtiter plates for performing the assay on the basis of preliminary testing. In two patients we also performed a cytotoxic assay using flow cytometry, where we dyed the target cells K562 with CFSE and 7-AAD dyes. Flow cytometry is a good alternative to LDH assay because we can determine the cytotoxicity on a single cell level with this technique. Our results mainly show large variations in NKA through the course of our clinical trial compared to our control values of samples taken before the first application of the trial substances, which is most likely a consequence of before mentioned therapy. Patients tested for NKA were representing three groups based on their responsiveness to antitumor therapy based on defined clinical parameters. We could not prove a correlation between NKA and clinical outcomes with our limited study subjects, but we observed trends which could in a large enough sample prove a correlation between the two. We would also need to design our clinical study for this purpose and take samples during a longer period of time, especially after the end of experimental treatment

Development of a novel in vitro cell model for evaluation of nanofiber mats immunogenicity

Immunological safety of nanofibers remains poorly reported within the scientific literature and lacks specific in vitro testing models distinct from those used to test nanoparticles. To address the challenges of currently used conventional setups being described in the literature, we developed a novel in vitro model for nanofiber mats immunogenicity testing, which enables standardization of tested surface area, excludes nanofiber mat edges, and ensures stable contacts of cells with nanofibers during the experiment. The effect of nanofibers was assessed on peripheral blood mononuclear cells (PBMCs) by measuring their metabolic activity using MTS cell proliferation assay, where key performance parameters, i.e. cell number, phytohemagglutinin-L (PHA-L) concentration, incubation time and cell lysis were optimized. Repeatability of results obtained with non-activated and PHA-L-activated PBMCs in contact with differently thick polycaprolactone nanofiber mats was compared using both models. Our model provided more reproducible results with lower variability, exhibiting its higher reliability and accuracy than the conventional one. Furthermore, results showed the presence of thicker mats resulted in reduced metabolic activity and PBMC proliferation without any observed cytotoxicity, providing additional insights into their non-immunogenic characteristics. The developed model enables more accurate biological assessment that can support new guidelines for in vitro nanofiber testing and formulation

Development of nanofibers with embedded liposomes containing an immunomodulatory drug using green electrospinning

Conventional treatments for chronic wounds are often ineffective, thus new therapeutic approaches are needed, such as the delivery of immunomodulatory drugs that can reduce inflammation, restore immune cell function, and facilitate tissue regeneration. A potential drug for such an approach is simvastatin, which has major drawbacks including poor solubility and chemical instability. With the aim of developing a dressing for wound healing, simvastatin and an antioxidant were incorporated into alginate/poly(ethylene oxide) nanofibers by green electrospinning without the use of organic solvents, thanks to their prior encapsulation into liposomes. The composite liposome–nanofiber formulations exhibited fibrillar morphology (160–312 nm) and unprecedentedly high phospholipid and drug content (76%). Transmission electron microscopy revealed dried liposomes as bright ellipsoidal spots homogeneously distributed over the nanofibers. After nanofiber hydration, the liposomes reconstituted in two size populations (~140 and ~435 nm), as revealed by cutting-edge MADLS^® analysis. Lastly, in vitro assays demonstrated that composite liposome–nanofiber formulations are superior to liposomal formulations due to a better safety profile in keratinocytes and peripheral blood mononuclear cells. Furthermore, both formulations exhibited similarly advantageous immunomodulatory effects, measured as decreased inflammation in vitro. A synergistic combination of the two nanodelivery systems shows promise for the development of efficient dressings for chronic wound treatment

Nanofibers with genotyped Bacillus strains exhibiting antibacterial and immunomodulatory activity

Biofilm-associated diseases such as periodontitis are widespread and challenging to treat which calls for new strategies for their effective management. Probiotics represent a promising approach for targeted treatment of dysbiosis in biofilm and modulation of host immune response. In this interdisciplinary study, nanofibers with two autochthonous Bacillus strains 27.3.Z and 25.2.M were developed. The strains were isolated from the oral microbiota of healthy individuals, and their genomes were sequenced and screened for genes associated with antimicrobial and immunomodulatory activities, virulence factors, and transferability of resistance to antibiotics. Spores of two Bacillus strains were incorporated individually or in combination into hydrophilic poly(ethylene oxide) (PEO) and composite PEO/alginate nanofibers. The nanofiber mats were characterised by a high loading of viable spores (> 7 log CFU/mg) and they maintained viability during electrospinning and 6 months of storage at room temperature. Spores were rapidly released from PEO nanofibers, while presence of alginate in the nanofibers prolonged their release. All formulations exhibited swelling, followed by transformation of the nanofiber mat into a hydrogel and polymer erosion mediating spore release kinetics. The investigated Bacillus strains released metabolites, which were not cytotoxic to peripheral blood mononuclear cells (PBMCs) in vitro. Moreover, their metabolites exhibited antibacterial activity against two periodontopathogens, an antiproliferative effect on PBMCs, and inhibition of PBMC expression of proinflammatory cytokines. In summary, the developed nanofiber-based delivery system represents a promising therapeutic approach to combat biofilm-associated disease on two fronts, namely via modulation of the local microbiota with probiotic bacteria and host immune response with their metabolites