Spontaneous regression of tumours. Possible cross reactivity of autoantibodies against carbonic anhydrase I.

Spontaneous tumour regression in patients after high dose therapy and autologous stem cell transplantation or patients with standard therapy is accompanied with the presence of high titers autoantibodies against carbonic anhydrase I (CA I). The concomitant presence of aplastic anaemia-like syndrome in these patients points to parallel bone marrow suppression during this period. It seems that CA I, an 'obscure' enzyme, does not have any significant physiological role in humans. One possible explanation points to the fact that autoantibodies against CA I may target another antigen(s) which is(are) important in tumour growth as well as in normal haematopoiesis. One of the candidates for such a target is the DNA polymerase theta.

Cryoprotective Mechanism of DMSO Induced by the Inhibitory Effect on Eutectic NaCl Crystallization.

Cryopreservation is a critical procedure in autologous hematopoietic stem cell transplantation. Dimethyl sulfoxide (DMSO) is the cryoprotectant of choice. Optimization of the cryopreservation protocol in the past revealed a dramatic loss of cell viability associated with a reduction of the DMSO concentration below 2 vol % in the freezing medium. The cryoprotective mechanism of DMSO is usually ascribed to the ability to suppress ice formation and reduce the adverse effects of the freeze-concentrated solution. This work proposes an alternative hypothesis considering the detrimental impact of NaCl eutectic crystallization on cell viability. Thermoanalytical and microstructural analysis of the DMSO effect on eutectic phase transformation of cryoprotective mixtures revealed a correlation between the loss of cell viability and eutectic NaCl crystallization. DMSO inhibits the eutectic crystallization of NaCl and preserves cell viability. Thermodynamic description of the inhibitory action and possible mechanism of cryoinjury are provided.

On crystallization of water confined in liposomes and cryoprotective action of DMSO.

In this work, the phase behavior of cryoprotective mixtures based on dimethyl sulfoxide (DMSO) mixed with a lipid bilayer consisting of dimyristoyl phosphatidylcholine (DMPC) was studied. This system represented a model of a biological cell and its membrane. The aim of the work was to clarify the origin of the cryoprotective action of low-concentrated mixtures (1-10 vol%) DMSO in water, representing mixtures used in cryopreservation in cell therapy. The combination of experimental techniques of differential scanning calorimetry (DSC) and positron annihilation lifetime spectroscopy (PALS) allowed a study of crystallization behavior of water confined in liposomes imitating the intracellular environment. The ability of liposomes to show the fundamental aspects of water phase behavior seen during freezing of biological cells was proved. The presence of an amorphous freeze-concentrated phase of DMSO in the frozen state was confirmed and its possible crystallization into the DMSO trihydrate and ice during thawing was demonstrated. Correlation between the critical temperature range for the loss of cell viability during slow thawing and the temperatures of freeze-concentrated phase crystallization was found. Based on this finding, possible mechanisms of DMSO cryoprotection are discussed with support brought by results for the studied model system. Quantification of the ice phase fraction in the frozen mixtures revealed that even low concentrations of DMSO can induce a considerable decrease in the amount of ice present.

Two new species of the genus Blattochaeta Reitter, 1910 (Insecta, Coleoptera, Leiodidae, Cholevinae, Leptodirini) from Montenegro.

Two further new species of the tribe Leptodirini Lacordaire, 1854 inhabit the subterranean world of the Southern Dinarides located in the northern and southeastern mountains of Montenegro: Blattochaeta brankojalzici D. Ceplík, Lakota J. Ceplík, sp. nov., and Blattochaeta peterhlavaci D. Ceplík, Lakota J. Ceplík, sp. nov. The new species are described, illustrated and dedicated to our dear friends in the field of subterranean biology and entomology. Morphological comparison with available related species, as well as the distribution, including a map for the whole genus Blattochaeta Reitter, 1910, and information on their natural history are given. A catalogue for the whole genus is provided and some taxonomic problems of related species are indicated.

Bathyscidius mikati (Udrzal, 1995), a new combination from the genus Bathyscia Schiødte, 1848 (Insecta, Coleoptera, Leiodidae, Cholevinae, Leptodirini).

The study of the type material as well as the additional specimens collected in the Rumija Mts., southeastern Montenegro revealed that Bathyscia mikati Udrzal, 1995 belongs to the genus Bathyscidius Jeannel, 1910. New combination, Bathyscidius mikati (Udrzal, 1995) comb. nov. is established. The species redescription, morphological comparison with related species, revised distribution, the map and the catalogue for the genus are provided.

Bathyscidius basarai, a new species of the tribe Leptodirini Lacordaire, 1854 (Insecta, Coleoptera, Leiodidae, Cholevinae) from Albania.

Bathyscidius basarai D. Ceplík, Lakota J. Ceplík, sp. nov., is described from Shpella e Blazit cave, located near the village of Bruç in central Albania. The new species is illustrated, morphological comparison with other species, as well as the distribution, including the map for the whole genus Bathyscidius Jeannel, 1910 and information on its natural history is given. The new species is tentatively placed in the subgenus Ionobathyscidius Polak Jalzic, 2019. Basic zoogeography for the subtribe Bathysiotina Guéorguiev, 1974, with the main focus on taxa of the Balkan Peninsula is provided.

Overexpression of CA1 mRNA and the CA I Protein in Tumor Cells Does Not Change the Gene Expression of the ECM Proteins.

In our study, we performed retroviral transduction to overexpress codon-optimized variant of gene encoding human carbonic anhydrase I (optiCA1) in two tumor cell lines PC3 and MDA-MB-231, derived from human prostatic and breast carcinoma respectively. We achieved significantly enhanced and stable overexpression of exogenous optiCA1 gene. The expression of endogenous, wild CA1 gene was found to be normally low (Ct 28.6 for PC3 cells) or below to the detection limit (Ct 35.5 for MDA-MB-231 cells). No morphological changes and no decreasing viability of tumor cells were observed upon stable overexpression of the optiCA1 gene. In our study we have shown that the overexpression of the optimized human CA1 in engineered PC3 and MDA-MB-231 cells did not induce similar changes as we observed in tumor cells cultivated in the presence of human sera containing extensively high titers of anti-CA I autoantibodies from patients with complete remission of malignant disease. In both optiCA1transduced cell lines, the expression of selected genes responsible for basal lamina assembly, cytoskeleton, extracellular matrix proteins and proto-oncogenes (COL1A1, COL4A4, LAMC2, CTHRC1, and WNT7B) was not changed.

Silencing of carbonic anhydrase I enhances the malignant potential of exosomes secreted by prostatic tumour cells.

We report results showing that the silencing of carbonic anhydrase I (siCA1) in prostatic (PC3) tumour cells has a significant impact on exosome formation. An increased diameter, concentration and diversity of the produced exosomes were noticed as a consequence of this knock-down. The protein composition of the exosomes' cargo was also altered. Liquid chromatography and mass spectrometry analyses identified 42 proteins significantly altered in PC3 siCA1 exosomes compared with controls. The affected proteins are mainly involved in metabolic processes, biogenesis, cell component organization and defense/immunity. Interestingly, almost all of them have been described as 'enhancers' of tumour development through the promotion of cell proliferation, migration and invasion. Thus, our results indicate that the reduced expression of the CA1 protein enhances the malignant potential of PC3 cells.

Microstructural free volume and dynamics of cryoprotective DMSO-water mixtures at low DMSO concentration.

This work investigates the free-volume properties of the dimethyl sulfoxide (DMSO)-water mixtures by positron annihilation lifetime spectroscopy over a wide temperature range of 20-320 K. The processes of melting and solidification of the water, DMSO and the DMSO-water mixtures at 1.8, 2.0 and 10% vol. DMSO respectively were studied. It was found that the recrystallization during heating of the water-DMSO cryoprotective mixtures above 160 K at low DMSO concentrations is affected by the amount of DMSO in the mixture. The amount of amorphous phase formed during cooling influences the hysteresis between cooling and heating cycles which could be crucial for cell survival. Experiments also show the time dependence of crystallization which indicates that rapid heating can suppress this secondary crystallization which is undesirable during the cell thawing process. Similar concentrations of DMSO (1.8% and 2% vol. DMSO in water) where a 2% vol. DMSO mixture secures cell survival but 1.8 vol% does not, showed differences in structural and dynamic properties that are key factors in cell survival. These results were supported by differential scanning calorimetry and low frequency dielectric spectroscopy measurements. The obtained data are in strong agreement with the observed cryoprotective efficacy of the DMSO-water mixtures on living cells.

Silencing of CA1 mRNA in tumour cells does not change the gene expression of the extracellular matrix proteins.

We report the silencing of CA1 mRNA in PC3 and MDA cells. The levels of mRNA coding CA1 protein in the knock-down mRNA (CA1 siRNA) cells have been measured by RT-PCR and were approximately 5% (PC3) and 20% (MDA-MB-231), respectively, of the level of control (Mock siRNA) used during silencing. In PC3 and MDA-MB-231 cells, the mRNAs for COL1A1 and COL4A4 were up-regulated. The mRNAs for CTHRC1, LAMC2, and WNT7B were not changed when compared to the control. The morphology of the cells during the treatments remained the same. On the Western blots, the lysate from the silenced cells showed lower levels of CA I as well.

Sera of patients with spontaneous tumour regression and elevated anti-CA I autoantibodies change the gene expression of ECM proteins.

Spontaneous tumour regression after high-dose therapy and autologous stem cell transplantation is associated with the aplastic anaemia-like syndrome and the presence of polyclonal autoantibodies against carbonic anhydrase I (CA I). When tumour cells were grown in vitro in the presence of patients' sera positive for anti-CA I autoantibodies, their morphological pattern was altered. These changes were accompanied by modifications in the gene expression profile. We observed downregulation of genes of the basal lamina assembly (collagen type IV alpha 4, the laminin subunit gamma 2), the extracellular matrix (collagen type I alpha 1), the cytoskeleton (keratin 14 type I), the collagen triple helix repeat containing 1 and the proto-oncogene WNT7B. On the other hand, the expression of the CA 1 gene was increased in the tumour cells. It was also noticed that the presence of anti-CA I autoantibodies did not impair tumour cell proliferation and cell viability in vitro. These findings were observed only in the presence of patients' sera positive for anti-CA I autoantibodies.

Studies on Adriaphaenops Noesske with the description of five new species from the Dinarides (Coleoptera: Carabidae: Trechini).

The species belonging to the trechine genus Adriaphaenops Noesske, 1928 are studied. As a result, seven currently known and five newly described species are recognized. Morphological characters of the habitus (especially the shape of head and pronotum) as well as male genitalia are widely used to delimit the species. The following new species are described: A. albanicus sp. nov. (Albania, Shkodër, Boga, Thatë Mts.), A. jasminkoi sp. nov. (Bosnia & Hercegovina, Nevesinje, Novakova pecina cave), A. mlejneki sp. nov. (Montenegro, Gornje Stravce, Kucke planine Mts.), A. njegosiensis sp. nov. (Montenegro, Cetinje, Cetinjska pecina cave) and A.rumijaensis sp. nov. (Montenegro, Virpazar, Rumija Mts.). Lectotype for A. stirni (Pretner, 1959) is designated. Data on the distribution and ecology of new taxa, complemented with descriptions of the type localities are provided. Key to the identification of all twelve Adriaphaenops species, as well as a key of all hitherto known aphaenopsoid Trechini genera from Dinarides are also given.

Endogean and cavernicolous Coleoptera of the Balkans. XVII. A new species of the genus Speluncarius Reitter, 1886 (Coleoptera, Carabidae, Harpalinae, Pterostichini) from Croatia.

The genus Speluncarius Reitter, 1886 (Harpalinae, Pterostichini) is made up by anophthalmous or almost blind species. The genus is related to Tapinopterus Schaum, 1858, which contains numerous species from the Balkan-Anatolian region and have more or less developed eyes. Speluncarius is distributed in the Western Palaearctic region; 26 species and one subspecies are known from Italy, the Balkan Peninsula and Turkey. The genus is classified in five subgenera, Elasmopterus Kraatz, 1886 (6 spp.); Hypogearius Jeannel, 1953 (3 spp.); Hypogium Tschitscherine, 1900 (1 sp.); Pontotapinus Guéorguiev & Lohaj, 2008 (1 sp.) and Speluncarius (s.str.) Reitter, 1886 (15 spp.) (Sciaky, 1982; Bousquet, 2003; Guéorguiev & Lohaj, 2008; Casale et al., 2013).

Synonymy of Reinholdina deelemanorum Giachino & Moravec, 2009 (Coleoptera: Leiodidae: Cholevinae: Leptodirini).

Reinholdina deelemanorum Giachino & Moravec, 2009 is a junior synonym of Parapropus neumanni Müller, 1911. The genus Reinholdina Giachino & Moravec, 2009 is placed in the synonymy with Parapropus Ganglbauer, 1899.

Overlap of epitopes recognized by anti-carbonic anhydrase I IgG in patients with malignancy-related aplastic anemia-like syndrome and in patients with aplastic anemia.

High titers of anti-carbonic anhydrase I (anti-CA I) autoantibodies were detected in the sera of patients with malignancies who developed an aplastic anemia-like (AA-like) syndrome after a high-dose therapy (HDT) and autologous stem cell transplantation (ASCT). It was found, that the presence of these anti-CA I autoantibodies is associated with spontaneous tumor regression. The main immunodominant epitopes of carbonic anhydrase isoform I (CA I) have previously been identified using epitope extraction technique in combination with mass spectrometric detection and bioinformatic verification. Similarly, the sera of patients with bona fide aplastic anemia (AA) who poorly responded to immunosuppressive treatment with anti-thymocyte globulin (ATG) demonstrated high titers of anti-CA I antibodies. In order to reveal differences between these antibodies, we applied the same methodology of epitope mapping procedure. Surprisingly, the anti-CA I antibodies from the both groups of patients compatibly recognized the same four candidate CA I epitopes--DGLAV, NVGHS, SLKPI, SSEQL. This finding may indicate common pathophysiological mechanisms in these two syndromes. However, at this moment it remains unresolved if anti-CA I antibodies are implicated in marrow or tumor suppression or are just an epi-phenomenon.