Cryopreservation Studies Of An Antifreeze Protein From The Desert Beetle Anatolica Polita

Antifreeze proteins (AFPs) are specialized evolutionary adaptations by certain cold climate organisms, including fish, plants, fungi, bacteria, and insects, to prevent cryo-injury. AFPs contribute to freeze resistance or tolerance by binding to the surface of ice crystals and preventing their growth, as well as inhibiting ice recrystallization (i.e., small crystals restructuring into large crystals) during large temperature fluctuations and constant sub-zero temperatures. AFPs have potential applications in cryopreservation and in the development of cryo-functional materials for medicine, the food industry, and agriculture. We investigated the ability of an antifreeze protein from the desert beetle Anatolica polita, ApAFP752, to confer cryoprotection in human embryonic kidney (HEK) 293T cells. HEK 293T cells were transfected with ApAFP752, and/or ApAFP752 was added to extracellular media prior to freezing at cryogenic temperature. AFP was assessed for its cryoprotective effects both intra- and extracellulary and both simultaneously at different concentrations with and without DMSO at different concentrations. After freezing and thawing, comparisons were made to DMSO or medium alone. ApAFP752 significantly improved cryopreserved cell survival when used with DMSO intracellularly. Extracellular ApAFP752 also significantly improved cryopreserved cell survival when included in the DMSO freezing medium. Intra- and extracellular ApAFP752 used together demonstrated the most significantly increased cryoprotection compared to DMSO alone at all DMSO concentrations. Together, these findings present potential methods for using an insect antifreeze protein to confer cryoprotection and improve the viability of cryopreserved mammalian cells

Solitary pulmonary amyloidoma mimicking lung cancer on 18F-FDG PET-CT scan in systemic lupus erythematosus patient

Localized amyloid deposits (tumoral amyloidosis or amyloidoma) are uncommon form of amyloidosis and nodular pulmonary amyloidomas are rarely found. This incidental finding can mimic a bronchopulmonary neoplasm and may occur secondarily to an infectious, inflammatory or lymphoproliferative disease. We report a case of a 62-year-old female with long-standing systemic lupus erythematosus (SLE) with low compliance who presented with radiologically-verified solitary pulmonary nodule. Work-up included positron emission tomography-computed tomography (PET-CT) scan, which revealed hypermetabolic uptake of (18)F-fluorodeoxyglucose, and lobectomy was performed. Staining of the tissue was positive for Congo red and was green birefringent under polarized light. Immunohistochemical methods excluded lymphoproliferative disease and confirmed amyloidoma. SLE was controlled with antimalarials and glucocorticoids. Pulmonary amyloidoma should be considered in the differential diagnosis of solitary lung nodules

Plućno krvarenje i glomerulonefritis s polumjesecima u pacijentu sa seropozitivnom antiglomerularnom bolesti bazne membrane i anti-neutrofilnim citoplazmičnim antitijelima

Anti-glomerular basement membrane (anti-GBM) disease is an acute and life-threatening systemic autoimmune disorder. The coexistence of circulating anti-neutrophil cytoplasmic antibodies (ANCA) and anti-GBM disease, the so-called double-positive disease (DPD), is exceptionally rare. We report a unique case of DPD manifesting as pulmonary-renal syndrome (PRS) in a 46-year-old woman who first presented with clinical and radiological suspicion of pneumonia. Chest computed tomography scan later revealed bilateral alveolar hemorrhage. Kidney biopsy showed necrotizing crescentic (100% glomeruli) glomerulonephritis. On immunofluorescence microscopy, glomeruli were global linear positive for IgG, confirming anti-GBM disease. Double positivity was detected for circulating anti-myeloperoxidase ANCA (p-ANCA) and anti-GBM antibodies. Acute renal failure evolved rapidly. Therapeutic plasma exchange (TPE) and hemodialysis (HD) were initiated early in combination with intravenous pulse corticosteroid therapy followed by oral methylprednisolone and cyclophosphamide. Pulmonary hemorrhage resolved, but renal function could not be preserved. The patient remains HD dependent. This case report highlights that pulmonary symptomatology may be the leading clinical presentation of PRS, with initially normal renal function at DPD onset. Early recognition and diagnosis are therefore crucial to timely clinical intervention. The role of prompt kidney biopsy and initiation of TPE and HD in PRS must not be underestimated.Bolest protiv glomerularne bazalne membrane (anti-GBM) je akutna i po život opasna sistemska autoimuna bolest. Pojava cirkulirajućih antitijela na citoplazmu granulocita (engl. anti-neutrophil cytoplasmic antibody, ANCA) u bolesnika sa anti-GBM glomerulonefritisom, tako zvana dvostruko pozitivna bolest (engl. double-positive disease, DPD), je vrlo rijetka. Prikazujemo 46 godišnju bolesnicu u koje je prvo klinički i radiološki postavljena sumnja na upalu pluća, a koja se kasnije manifestirala sa pulmo-renalnim sindromom (PRS) odnosno DPD. Kompjutorskom tomografijom pluća dokazano je alveolarno krvarenje. Biopsijom bubrega dokazan je nekrotizirajući glomerulonefritis (100 % glomerula). Imunoflorescencija je pokazala pozitivne linearne IgG depozite, što odgovara anti-GBM glomerulonefritisu. U bolesnice su dokazana antitijela na mijeloperoksidazu p-ANCA i antitijela na glomerularnu bazalnu membranu. Liječena je terapijskom izmjenom plazme (engl. therapeutic plasma exchange, TPE), hemodijalizom te kombinacijom parenteralne pulsne terapije kortikosteroidima, kasnije oralnom primjenom metilprednisolona i ciklofosfamida. Došlo je do regresije krvarenja u plućima ali se bubrežna funkcija nije oporavila, zbog čega smo nastavili s redovitim hemodijalizama. Ovaj prikaz bolesnice pokazuje kako u DPD, plućna simptomatologija može biti vodeći simptom PRS sa urednom bubrežnom funkcijom u početku. Rano prepoznavanja i dijagnoza su značajni za pravovremeni početak liječenja. Potrebno je naglasiti značaj rane biopsije bubrega, ranog početka TPE te po potrebi i nadomještanje bubrežne funkcije hemodijalizom

Lažno pozitivna fenotipska detekcija metalo-beta-laktamaza u Acinetobacter Baumannii

Phenotypic detection of metallo-β-lactamases (MBLs) in Acinetobacter (A.) baumannii is a serious challenge to clinical microbiologists. MBLs are inhibited by metal chelators such as ethylenediaminetetraacetic acid) (EDTA). Production of MBLs cannot be recognized based on resistance phenotype. Therefore, phenotypic tests using EDTA are recommended. The aim of this study was to investigate the sensitivity and specificity of inhibitor based tests (EDTA) for detection of MBL. A total of 172 A. baumannii strains (123 carbapenemase positive and 49 carbapenemase negative) were analyzed. Phenotypic detection of MBLs was performed by the combined disk test with EDTA (CDT-EDTA) and EPI-dilution test (EPI-DT). Both tests were positive in all 11 isolates possessing VIM-1 MBL, showing 100% sensitivity. However, false positive results were observed in strains with class D carbapenemases using both tests, i.e. all OXA-23 and OXA-24/40 producing organisms and most OXA-58 positive strains (77% with CDT-EDTA vs. 65% with EPI-DT). False positive results can occur because oxacillinases are converted to a less active state in the presence of EDTA, leading to augmentation of the inhibition zone around the carbapenem disk or reduction of carbapenem minimum inhibitory concentrations. This study showed high sensitivity but low specificity of phenotypic methods in the detection of MBLs.Fenotipska detekcija metalo-β-laktamaza (MBL) u Acinetobacter (A.) baumannii je velik izazov kliničkim mikrobiolozima. MBL su inhibirane metalnim kelatorima kao što je etilendiamin tetraoctena kiselina (EDTA). Produkcija MBL ne može se prepoznati na temelju fenotipa rezistencije. Zbog toga se preporuča primjena fenotipskih testova koji rabe metalne kelatore kao što je EDTA. Cilj istraživanja bio je utvrditi osjetljivost i specifičnost testova s inhibitorom (EDTA) u otkrivanju MBL u A. baumannii. Analizirana su 172 izolata A. baumannii (123 karbapenemaza pozitivnih i 49 karbapenemaza negativnih). Fenotipska detekcija MBL je provedena metodom kombiniranih diskova (EDTA CDT-EDTA) i EPI-dilucijskom metodom (EPI-DT). Oba testa su bila pozitivna u svih 11 izolata pozitivnih na VIM-1 MBL, što odgovara osjetljivosti od 100%. Lažno pozitivni rezultati su dobiveni u karbapenem-rezistentnim izolatima pozitivnim na klasu D karbapenemaza u oba testa: svi OXA-23 i OXA-24/40 kao i većina OXA-58 pozitivnih sojeva (77% u CDT-EDTA i 65% u EPI-DT). Lažno pozitivni rezultati u testovima s inhibitorom nastaju zbog toga što oksacilinaze prelaze u stanje manje aktivnosti u prisustvu EDTA, što dovodi do smanjenja minimalne inhibitorne koncentracije karbapenema ili do uvećanja zone oko diska karbapenema. Studija je pokazala visoku osjetljivost, ali nisku specifičnost fenotipskih testova za dokaz MBL u A. baumannii, što ukazuje na neophodnost korištenja molekularnih metoda

A subset of non-small cell lung cancer patients treated with pemetrexed show 18f-fluorothymidine ‘flare’ on positron emission tomography

Thymidylate synthase (TS) remains a major target for cancer therapy. TS inhibition elicits increases in DNA salvage pathway activity, detected as a transient compensatory “flare” in 3′-deoxy-3′-[18F]fluorothymidine positron emission tomography (18F-FLT PET). We determined the magnitude of the 18F-FLT flare in non-small cell lung cancer (NSCLC) patients treated with the antifolate pemetrexed in relation to clinical outcome. Method: Twenty-one patients with advanced/metastatic non-small cell lung cancer (NSCLC) scheduled to receive palliative pemetrexed ± platinum-based chemotherapy underwent 18F-FLT PET at baseline and 4 h after initiating single-agent pemetrexed. Plasma deoxyuridine (dUrd) levels and thymidine kinase 1 (TK1) activity were measured before each scan. Patients were then treated with the combination therapy. The 18F-FLT PET variables were compared to RECIST 1.1 and overall survival (OS). Results: Nineteen patients had evaluable PET scans at both time points. A total of 32% (6/19) of patients showed 18F-FLT flares (>20% change in SUVmax-wsum). At the lesion level, only one patient had an FLT flare in all the lesions above (test–retest borders). The remaining had varied uptake. An 18F-FLT flare occurred in all lesions in 1 patient, while another patient had an 18F-FLT reduction in all lesions; 17 patients showed varied lesion uptake. All patients showed global TS inhibition reflected in plasma dUrd levels (p < 0.001) and 18F-FLT flares of TS-responsive normal tissues including small bowel and bone marrow (p = 0.004 each). Notably, 83% (5/6) of patients who exhibited 18F-FLT flares were also RECIST responders with a median OS of 31 m, unlike patients who did not exhibit 18F-FLT flares (15 m). Baseline plasma TK1 was prognostic of survival but its activity remained unchanged following treatment. Conclusions: The better radiological response and longer survival observed in patients with an 18F-FLT flare suggest the efficacy of the tracer as an indicator of the early therapeutic response to pemetrexed in NSCLC

Plasma Brain-Derived Neurotrophic Factor (BDNF) Concentration and BDNF/TrkB Gene Polymorphisms in Croatian Adults with Asthma

Brain-derived neurotrophic factor (BDNF) and its tropomyosin-related kinase B (TrkB) receptor might contribute to normal lung functioning and immune responses ; however, their role in asthma remains unclear. Plasma BDNF concentrations, as well as BDNF and NTRK2 (TrkB gene) polymorphisms, were investigated in 120 asthma patients and 120 healthy individuals using enzyme-linked immunosorbent assay and polymerase chain reaction, respectively. The genotype and allele frequencies of BDNF Val66Met (rs6265) and NTRK2 rs1439050 polymorphisms did not differ between healthy individuals and asthma patients, nor between patients grouped according to severity or different asthma phenotypes. Although plasma BDNF concentrations were higher among healthy subjects carrying the BDNF Val66Met GG genotype compared to the A allele carriers, such differences were not detected in asthma patients, suggesting the influences of other factors. Plasma BDNF concentration was not affected by NTRK2 rs1439050 polymorphism. Asthma patients had higher plasma BDNF concentrations than control subjects ; however, no differences were found between patients subdivided according to asthma severity, or Type-2, allergic, and eosinophilic asthma. Higher plasma BDNF levels were observed in asthma patients with aspirin sensitivity and aspirin-exacerbated respiratory disease. These results suggest that plasma BDNF may serve as a potential peripheral biomarker for asthma, particularly asthma with aspirin sensitivity