The Ubiquitin-Associated and SH3 Domain-Containing Proteins (UBASH3) Family in Mammalian Development and Immune Response.

UBASH3A and UBASH3B are protein families of atypical protein tyrosine phosphatases that function as regulators of various cellular processes during mammalian development. As UBASH3A has only mild phosphatase activity, its regulatory effects are based on the phosphatase-independent mechanisms. On the contrary, UBASH3B has strong phosphatase activity, and the suppression of its receptor signalling is mediated by Syk and Zap-70 kinases. The regulatory functions of UBASH3A and UBASH3B are particularly evident in the lymphoid tissues and kidney development. These tyrosine phosphatases are also known to play key roles in autoimmunity and neoplasms. However, their involvement in mammalian development and its regulatory functions are largely unknown and are discussed in this review.

Expression Patterns of Serotonin Receptors 5-HT1A, 5-HT2A, and 5-HT3A during Human Fetal Lung Development.

We analyzed the expression of the serotonin receptors 5-HT1A, 5-HT2A, and 5-HT3A at four different stages of fetal lung development from 12 to 40 weeks of gestation, divided into four groups: the pseudoglandular stage (12-16th week of development; n = 8), the canalicular stage (16th-26th week of development; n = 7), the saccular stage (26th-36th week of development; n = 5), and the alveolar stage (36th-40th week of development; n = 5). The strongest expression of all three receptor types was found in the epithelium of the proximal airways during the pseudoglandular, canalicular, and saccular stages and in a vascular wall. 5-HT1A was also strongly expressed in the smooth muscle cells of the proximal airway. Vascular smooth muscle cells and endothelium occasionally showed a strong expression of 5-HT1A and 5-HT2A. In the alveolar stage, the expression of 5-HT1A, 5-HT2A, and 5-HT3A was detected in both type I (p1) and type II (p2) pneumocytes, with a stronger expression in p2. A significant decrease in percent the 5-HT2A area and in the integrated density was observed at the alveolar stage. On the other hand, a significant decrease in the percentage area but an increase in the integrated density was observed for 5-HT3A toward the alveolar stage, suggesting that a smaller number of cells expressed 5-HT3A but that they (p1 and p2) significantly increased their 5-HT3A expression at the alveolar stage. The results presented provided us with new data on the development and function of the serotonin system in the human fetal lung and gave us insight into their possible involvement in the pathogenesis of lung pathology, particularly that characteristic of the neonatal period.

Expression Pattern of Sonic Hedgehog, Patched and Smoothened in Clear Cell Renal Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the deadliest neoplasm of the urinary tract, and we are still far from completely understanding ccRCC development and treatment. The renal tissue paraffin blocks (20) of patients with ccRCC were collected at the University Hospital in Split from 2019 to 2020, and tissue sections were stained with patched (PTCH), anti-smoothened (SMO) and anti-Sonic Hedgehog (SHH) antibodies. SHH was highly expressed (31.9%) in grade 1 tumour, it being higher than all other grades and the control (p < 0.001-p < 0.0001). The trend of a linear decrease in the expression of SHH was observed with the progression of the tumour grade (p < 0.0001). PTCH expression was significantly lower in grades 1 and 2 in comparison to the control (p < 0.01) and grade 4 (p < 0.0001). A significant increase in the expression of SMO was found in grade 4 compared to all other grades (p < 0.0001) and the control (p < 0.001). The strong expression of SHH was observed in carcinoma cells of the G1 stage with a diffuse staining pattern (>50% of neoplastic cells). Stroma and/or inflammatory infiltrate display no staining and no expression of SHH in G1 and G2, while mild focal staining (10-50% of neoplastic cells) was observed in G3 and G4. Patients with high PTCH and low SMO expression had significant time survival differences (p = 0.0005 and p = 0.029, respectively). Therefore, high levels of PTCH and low levels of SMO expression are important markers of better survival rates in ccRCC patients.

Expression of Cell Cycle Markers and Proliferation Factors during Human Eye Embryogenesis and Tumorigenesis.

The expression pattern of the markers p19, Ki-67, MSX1, MSX2, PDL1, pRB, and CYCLINA2 was quantitatively and semiquantitatively analyzed in histologic sections of the developing and postnatal human eye at week 8, in retinoblastoma, and in various uveal melanomas post hoc studies by double immunofluorescence. The p19 immunoreactivity characterized retinal and/or choroidal cells in healthy and tumor tissues: expression was lower in the postnatal retina than in the developing retina and retinoblastoma, whereas it was high in epithelioid melanomas. Ki67 expression was high in the developing eye, retinoblastoma, and choroidal melanomas. MSX1 and MSX2 expression was similar in the developing eye and retinoblastoma, whereas it was absent in the postnatal eye. Their different expression was evident between epithelioid and myxoid melanomas. Similarly, PDL1 was absent in epithelioid melanomas, whereas it was highly expressed in developing and tumor tissues. Expression of pRB and CYCA2 was characteristic of developing and tumorous eye samples but not of the healthy postnatal eye. The observed expression differences of the analyzed markers correlate with the origin and stage of cell differentiation of the tissue samples. The fine balance of expression could play a role in both human eye development and ocular tumorigenesis. Therefore, understanding their relationship and interplay could open new avenues for potential therapeutic interventions and a better understanding of the mechanisms underlying the developmental plasticity of the eye and the development of neoplasms.

The Expression of Connexin 37, 40, 43, 45 and Pannexin 1 in the Early Human Retina and Choroid Development and Tumorigenesis.

The expression pattern of Connexins (Cx) 37, 40, 43, 45 and Pannexin 1 (Pnx1) was analyzed immunohistochemically, as well as semi-quantitatively and quantitatively in histological sections of developing 8th- to 12th-week human eyes and postnatal healthy eye, in retinoblastoma and different uveal melanomas. Expressions of both Cx37 and Cx43 increased during development but diminished in the postnatal period, being higher in the retina than in the choroid. Cx37 was highly expressed in the choroid of retinoblastoma, and Cx43 in epitheloid melanoma, while they were both increasingly expressed in mixoid melanoma. In contrast, mild retinal Cx40 expression during development increased to strong in postnatal period, while it was significantly higher in the choroid of mixoid melanoma. Cx45 showed significantly higher expression in the developing retina compared to other samples, while it became low postnatally and in all types of melanoma. Pnx1 was increasingly expressed in developing choroid but became lower in the postnatal eye. It was strongly expressed in epithelial and spindle melanoma, and particularly in retinoblastoma. Our results indicate importance of Cx37 and Cx40 expression in normal and pathological vascularization, and Cx43 expression in inflammatory response. Whereas Cx45 is involved in early stages of eye development, Pnx1might influence cell metabolism. Additionally, Cx43 might be a potential biomarker of tumor prognosis.

Immunofluorescence Analysis of NF-kB and iNOS Expression in Different Cell Populations during Early and Advanced Knee Osteoarthritis.

Synovitis of the knee synovium is proven to be a precursor of knee osteoarthritis (OA), leading to a radiologically advanced stage of the disease. This study was conducted to elucidate the expression pattern of different inflammatory factors-NF-kB, iNOS, and MMP-9 in a subpopulation of synovial cells. Thirty synovial membrane intra-operative biopsies of patients (ten controls, ten with early OA, and ten with advanced OA, according to the Kellgren-Lawrence radiological score) were immunohistochemically stained for NF-kB, iNOS, and MMP9, and for different cell markers for macrophages, fibroblasts, leukocytes, lymphocytes, blood vessel endothelial cells, and blood vessel smooth muscle cells. The total number of CD68+/NF-kB+ cells/mm2 in the intima of early OA patients (median = 2359) was significantly higher compared to the total number of vimentin+/Nf-kB+ cells/mm2 (median = 1321) and LCA+/NF-kB+ cells/mm2 (median = 64) (p < 0.001 and p < 0.0001, respectively). The total number of LCA+/NF-kB+ cells/mm2 in the subintima of advanced OA patients (median = 2123) was significantly higher compared to the total number of vimentin+/NF-kB+ cells/mm2 (median = 14) and CD68+/NF-kB+ cells/mm2 (median = 29) (p < 0.0001). The total number of CD68+/iNOS+ cells/mm2 in the intima of both early and advanced OA patients was significantly higher compared to the total number of vimentin+/iNOS+ cells/mm2 and LCA+/iNOS+ cells/mm2 (p < 0.0001 and p < 0.001, respectively). The total number of CD68+/MMP-9+ cells/mm2 in the intima of both early and advanced OA patients was significantly higher compared to the total number of vimentin+/MMP-9+ cells/mm2 and CD5+/MMP-9+ cells/mm2 (p < 0.0001). Macrophages may have a leading role in OA progression through the NF-kB production of inflammatory factors (iNOS and MMP-9) in the intima, except in advanced OA, where leukocytes could have a dominant role through NF-kB production in subintima. The blocking of macrophageal and leukocyte NF-kB expression is a possible therapeutic target as a disease modifying drug.

Expression Pattern of iNOS, BCL-2 and MMP-9 in the Hip Synovium Tissue of Patients with Osteoarthritis.

Hip osteoarthritis (HOA) is characterized by degradation of the cartilage and synovitis. However, the pathohistological effects of synovial tissue inflammation on HOA are not clear. The aim of this study was to evaluate the expression of iNOS, BCL-2 and MMP-9 markers in different synovial cell populations. A total of 32 patients were evaluated retrospectively. Age, sex, height, weight, body mass index were recorded and lymphocyte, fibrocytes and macrophages were analysed in tissue sections. Osteoarthritis cartilage histopathology assessment system (OARSI), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Krenn score, Harris Hip Score (HHS) and Kellgren-Lawrence (K-L) grading of the hip joints were performed. Total hip arthroplasty was performed on 32 patients and controls. Patients were divided into two groups according to their disease severity. The tissues were immunohistochemically analysed. K-L grade and Krenn score differ between all three groups, but also between moderate and severe OA. Synovial lining cell layer, resident cells in stroma and especially inflammatory infiltration were increasing with severity of OA. iNOS expression in both intima and subintima was positively correlated with Krenn score in moderate and severe osteoarthritis (OA) groups. Expression of BCL-2 in intima of severe OA patients was positively correlated with Krenn score. In conclusion, iNOS, BCL-2 and MMP-9 are involved in the regulation of HOA. Our study indicates a relationship between the pathohistological features, the synovial inflammation and the cartilage condition at the time of hip replacement due to OA or femoral neck fracture.

Expression and localization of DAB1 and Reelin during normal human kidney development.

AIM: To explore the spatial and temporal expression patterns of DAB1 and Reelin in the developing and postnatal healthy human kidneys as potential determinants of kidney development. METHODS: Paraffin-embedded fetal kidney tissue between the 13/14th and 38th developmental weeks (dw) and postnatal tissue at 1.5 and 7 years were stained with DAB1 and Reelin antibodies by double immunofluorescence. RESULTS: During the fetal kidney development and postnatal period, DAB1 and Reelin showed specific spatial expression pattern and diverse fluorescence intensity. During the fetal period, DAB1 was strongly expressed in the distal convoluted tubules (DCT), with strong reactivity, and diversely in the proximal convoluted tubules (PCT) and glomeruli. In the postnatal period, DAB1 expression decreased. The strongest Reelin expression in early fetal stages was observed in the PCT. In the postnatal period, Reelin expression decreased dramatically in all observed structures. These two markers were colocalized during early developmental stages, mostly in PCT, DCT, and podocytes. CONCLUSION: The appearance of DAB1 and Reelin during fetal kidney development confirms their potential significant role in the formation of kidney structure or function. High DAB1 expression in the DCT implies its regulatory role in tubular formation or function maintenance during development. Reelin was highly expressed in human kidneys at early fetal stages, mostly in the PCT, while at later fetal stages and postnatal period its expression decreased.

Correlation of anxiety and chronic pain to grade of synovitis in patients with knee osteoarthritis.

BACKGROUND: This study was conducted with the objective of finding out the correlation between synovial inflammation measured histopathologically and subjective symptoms; anxiety and chronic pain, in knee osteoarthritis (OA). SUBJECTS AND METHODS: Thirty patients were included in the study. Ten of them were in a control group with meniscal injury, ten had early OA and 10 had late OA. Knee radiographs were graded using Kellgren-Lawrence classification. Synovial biopsies were taken during surgery or arthroscopy and synovitis score was measured by Krenns method. Anxiety was measured with Beck Anxiety Inventory and pain was taken as part of the WOMAC score (The Western Ontario and McMaster Universities Arthritis Index). RESULTS: Krenn synovitis score was determined as: no synovitis, low-grade synovitis and high-grade synovitis. Group with low-grade synovitis had significantly higher pain score than high-grade synovitis group (p=0.011). No-synovitis group had significantly lower Beck Anxiety Inventory than low-grade synovitis group (p=0.014) and high-grade synovitis (p=0.008). There are no significant differences between low-grade synovitis and high-grade synovitis in anxiety score (p=0.912). CONCLUSIONS: Chronic pain is more present in late osteoarthritis, when synovitis is less pronounced. Anxiety affects patients who suffer osteoarthritis, but it is statistically the same regarding synovitis grade, i.e. whether it is early or late osteoarthritis.

Kappa Free Light Chains in Cerebrospinal Fluid of Patients with Identified Oligoclonal Immunoglobulin G.

BACKGROUND: Production of kappa free light chains (KFLC) represents a part of humoral immune response, along with the synthesis of intrathecal immunoglobulins. Increased concentrations of immunoglobulin G light chains, kappa and lambda chains, were identified through research of numerous diseases of central nervous system. The qualitative method of isoelectric focusing (IEF) followed by immunofixation currently represents the accepted standard in identifying oligoclonal bands (OCB), but establishing a sensitive immunonephelometric method for quantification of kappa free light chains (KFLC) in cerebrospinal fluid (CSF) has paved a way for new diagnostic possibilities. Andersson classified the pattern types of OCB, ranging from type 1 to type 5, wherein types 2 and 3 indicate intrathecal synthesis. Our aim was to determine KFLC in CSF of patients with clinically isolated syndrome (CIS) who had presented with type 2 and type 3 OCB, to determine if there is a difference in concentrations between those two groups and to establish a borderline value of KFLC which would enable differential diagnostics. SUBJECTS AND METHODS: 70 patients, who underwent lumbar punction for CSF analysis and had their blood sampled through the cubital vein, participated in the study. Patients were classified according to Andersson as type 2 or type 3, which besides adulthood, represented the inclusion criteria. The average age of patients classified as type 2 was 36 years, and those classified as type 3 was 39 years, where it is evident that there was not a statistically significant difference (p=0.0685). We used a qualitative electrophoretic technique of IEF with agarose gel followed by immunofixation, and a quantitative immunonephelometric method. All results were interpreted on a level of statistic significance of p<0.05. RESULTS: CSF KFLC concentrations in type 3 were statistically and significantly elevated with regard to type 2 (Mann-Whitney test, p=0.0430). The median for KFLC in type 2 was 0.9 mg/L, while the median for KFLC in type 3 was 2.71 mg/L, and the detection limit for both types was 0.18 mg/L. We used a statistical ROC curve to determine that KFLC concentration can be used for differential diagnostics, meaning it can discriminate type 2 from type 3 with clinical sensitivity of 61% and clinical specificity of 71% (AUC=0.641) (p=0.037). CONCLUSION: Despite the obtained statistically significant differences in concentrations of KFLC between types of OCBs and ROC analysis results, determination of KFLC by a nephelometric method, insufficiently strong clinical sensitivity and specificity does not justify abandonment of IEF method followed by immunofixation.

Diagnostic Value of Fine Needle Aspiration Cytology for Breast Tumors

Breast cancer is the most frequently diagnosed cancer and the leading cause of death from cancer in women. The accuracy of diagnosis can be increased with a combination of clinical examination, imaging diagnostics, and fi ne needle aspiration cytology (FNAC) or core needle biopsy, also known as triple test. The aim of the study was to evaluate the sensitivity and specificity of FNAC in the diagnosis of breast tumors in our institution by correlating it with histopathology findings. We assessed the accuracy of 124 FNAC findings by comparing cytological diagnosis of breast masses with the diagnoses from histopathology reports obtained by surgery. Statistical analysis showed 95.1% accuracy, 97.7% sensitivity, 89.1% specificity, 95.5% positive predictive value and 94.2% negative predictive value of FNAC. Study results indicated that FNAC could be used as a highly reliable tool in the differential diagnosis of breast tumors, in combination with clinical and imaging findings, especially in developing countries with limited financial resources.

Effect of Granzyme K, FasL and Interferon-γ Expression in Placentas with Preeclampsia.

This study aimed to investigate the cytotoxic activity of decidual lymphocytes and the mRNA/protein expression of cytotoxic proteins in various cell types in the context of preeclampsia (PE) compared to those of healthy pregnancies. We analyzed fresh decidua basalis tissue and tissue embedded in paraffin (FFPE) from PE pregnancies (n = 15) and compared them with those of healthy pregnancies (n = 15) of the corresponding gestational age. Using double immunofluorescence staining, we observed differences in the intensity and distribution of staining for granzyme K (GZMK) and FasL in extravillous trophoblasts. RT-qPCR analysis of FFPE placental tissue showed that GZMK mRNA expression was statistically higher (p < 0.0001) in PE compared to that of healthy controls. On the contrary, there was a low expression (p < 0.001) of FasL mRNA in PE compared to controls, while there was no statistically significant difference for IFN-γ mRNA between PE and controls. Although the level of cytotoxic activity changed depending on the ratio of effector and target cells, there was no significant difference observed between PE and controls in this in vitro study. In conclusion, in PE, extravillous trophoblasts exhibited increased expression of GZMK and decreased expression of FasL. These changes may contribute to impaired trophoblast invasion. However, these alterations did not appear to affect the cytotoxic properties of decidual lymphocytes. Additionally, the possibility of cell sorter separation of decidual lymphocytes would greatly contribute to a better understanding of single cells' genetic profiles.

Expression of IMP3 and LIN28A RNA-Binding Proteins in Placentas of Patients with Pre-Eclampsia with and without Severe Features.

BACKGROUND: this study aimed to determine the expression of RNA-binding oncofetal proteins IMP3 and LIN28A in extravillous (EVT) and villous trophoblast (VT) cells of placentas from pre-eclamptic (PE) pregnancies to better understand the pathogenesis of PE. METHODS: placental tissue of 10 patients with PE with severe features, 10 patients with PE without severe features and 20 age-matched healthy pregnancy controls were analyzed by immunohistochemistry, double immunofluorescence and qPCR. RESULTS: We found a decreased percentage of IMP3-positive EVT cells in PE with and without severe features compared to that of the healthy control (p < 0.001). IMP3 expression was significantly low in VT of PE placentas compared to that of the healthy control (p = 0.002). There was no significant difference in LIN28A expression between groups of PE and the control group. Additionally, we noticed the trend toward downregulation of IMP3 mRNA and LIN28A mRNA in severe PE compared to that of healthy controls. CONCLUSIONS: We demonstrated that IMP3 expression is decreased in EVT and VT cells of placentas from pregnancies complicated with both PE with and without severe features. However, additional functional investigations are needed to clarify the role of IMP3 as a potential therapeutic target in the management of PE.

Clinical Significance and Expression Pattern of RIP5 and VGLL4 in Clear Cell Renal Cell Carcinoma Patients Treated with Sunitinib.

While clear cell renal cell carcinoma (ccRCC) is curable, advanced metastatic (mRCC) remains a clinical challenge. We analyzed clinical, pathohistological, and molecular data (Receptor Interacting Protein 5-RIP5 and Vestigial Like Family Member 4-VGLL4 expression) of 55 mRCC patients treated with first-line treatment with sunitinib. The trend of linear increase in the protein expression of RIP5 was observed with the progression of tumor grade. Overall, 80% of RIP5-positive cells were in the control kidneys and high-grade mRCC. On the contrary, RIP5 displayed low expression in grade 2 mRCC (5.63%). The trend of linear decrease in the expression of VGLL4 was observed with the progression of tumor grade. The highest protein expression of VGLL4 was observed in grade 2 (87.82%) in comparison to grade 3 and 4 and control. High expression of RIP5 mRNA was associated with longer first-line overall survival and longer progression-free survival in mRCC. In addition, a high VGLL4 mRNA expression showed better overall survival in patients with ccRCC. In conclusion, high mRNA expression of RIP5 and VGLL4 are important markers of better survival rates in mRCC patients.

Spatio-Temporal Expression Pattern of CAKUT Candidate Genes DLG1 and KIF12 during Human Kidney Development.

We aimed to investigate expression of the novel susceptibility genes for CAKUT, DLG1 and KIF12, proposed by a systematic in silico approach, in developing and postnatal healthy human kidneys to provide information about their spatiotemporal expression pattern. We analyzed expression of their protein products by immunohistochemistry and immunofluorescence and quantified relative mRNA levels by RT-qPCR. Statistically significant differences in expression patterns were observed between certain developmental stages. Strong expression of DLG1 was observed in the developing kidney, with a gradual decrease from the first phase of kidney development (Ph1) until the third phase (Ph3), when most nephrons are formed; at later stages, the highest expression was observed in the tubules. KIF12 was highly expressed in the developing structures, especially in Ph1, with a gradual decrease until the postnatal phase, which would indicate a significant role in nephrogenesis. Co-localization of DLG1 and KIF12 was pronounced in Ph1, especially on the apical side of the tubular epithelial cells. Thereafter, their expression gradually became weaker and was only visible as punctate staining in Ph4. The direct association of DLG1 with KIF12 as control genes of normal kidney development may reveal their new functional aspect in renal tubular epithelial cells.

Metformin and Thymoquinone Synergistically Inhibit Proliferation of Imatinib-Resistant Human Leukemic Cells.

Chemotherapy resistance is one of the major challenges in cancer treatment, including leukemia. A massive array of research is evaluating combinations of drugs directed against different intracellular signaling molecules to overcome cancer resistance, increase therapy effectiveness, and decrease its adverse effects. Combining chemicals with proven safety profiles, such as drugs already used in therapy and active substances isolated from natural sources, could potentially have superior effects compared to monotherapies. In this study, we evaluated the effects of metformin and thymoquinone (TQ) as monotherapy and combinatorial treatments in chronic myeloid leukemia (CML) cell lines sensitive and resistant to imatinib therapy. The effects were also evaluated in primary monocytic acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) cells. Both compounds induced a dose- and time-dependent decrease of viability and proliferation in tested cells. Metformin had similar IC50 values in imatinib-sensitive and imatinib-resistant cell lines. IC50 values of TQ were significantly higher in imatinib-resistant cells, but with a limited resistance index (2.4). Synergistic effects of combinatorial treatments were observed in all tested cell lines, as well as in primary cells. The strongest synergistic effects were observed in the inhibition of imatinib-resistant cell line proliferation. Metformin and TQ inhibited the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and induced apoptosis in tested cell lines and primary cells. The enhanced effects of combinatorial treatments on the induction of apoptosis were more dominant in imatinib-resistant compared to imatinib-sensitive CML cells. Primary cells were more sensitive to combinatorial treatments compared to cell lines. A combination of 1.25 mM metformin and 0.625 µM TQ increased the levels of cleaved poly (ADP-ribose) polymerase (PARP), decreased the levels of proliferation regulatory proteins, and inhibited protein kinase B (Akt) and NF-κB signaling in primary CLL cells. This study demonstrates that combinatorial treatments of imatinib-resistant malignant clones with metformin and TQ by complementary intracellular multi-targeting represents a promising approach in future studies.

The Interplay of Cx26, Cx32, Cx37, Cx40, Cx43, Cx45, and Panx1 in Inner-Ear Development of Yotari (dab1-/-) Mice and Humans.

We investigated DAB1-protein deficiency in the inner-ear development of yotari in comparison to humans and wild-type (wt) mice by immunofluorescence for the expression of connexins (Cxs) and the pannexin Panx1. The spatial and temporal dynamics of Cx26, Cx32, Cx37, Cx40, Cx43, Cx45, and Panx1 were determined in the sixth and eighth weeks of human development and at the corresponding mouse embryonic E13.5 and E15.5, in order to examine gap junction intercellular communication (GJIC) and hemichannel formation. The quantification of the area percentage covered by positive signal was performed for the epithelium and mesenchyme of the cochlear and semicircular ducts and is expressed as the mean ± SD. The data were analysed by one-way ANOVA. Almost all of the examined Cxs were significantly decreased in the cochlear and semicircular ducts of yotari compared to wt and humans, except for Cx32, which was significantly higher in yotari. Cx40 dominated in human inner-ear development, while yotari and wt had decreased expression. The Panx1 expression in yotari was significantly lower than that in the wt and human inner ear, except at E13.5 in the mesenchyme of the wt and epithelium and mesenchyme of humans. Our results emphasize the relevance of GJIC during the development of vestibular and cochlear functions, where they can serve as potential therapeutic targets in inner-ear impairments.

Expression and localization of FGFR1, FGFR2 and CTGF during normal human lung development.

Aim of our study was to provide insight into the temporal and spatial expression of FGFR1, FGFR2 and CTGF during normal human lung development which may have an important impact on understanding occurrence of developmental lung anomalies. Morphological parameters were analysed using double immunofluorescence on human embryonal (6th and 7th developmental week-dw) and foetal (8th, 9th and 16th developmental week) human lung samples. FGFR1 and FGFR2 was positive during all the dw in both the epithelium and mesenchyme. The highest number of FGFR1 positive cells was observed during the 6th dw (112/mm2) and 9th dw (87/mm2) in the epithelium compared to the 7th, 8th and 16th dw (Kruskal-Wallis test, p < 0.001, p < 0.0001). The highest number of FGFR1 positive cells in the mesenchyme was observed during the 8th dw (19/mm2) and 16th dw (13/mm2) compared to the 6th, 7th, and 9th dw (Kruskal-Wallis test, p < 0.001, p < 0.0001). The number of FGFR1 positive cells in the epithelium was higher for FGFR2 compared to number of positive cells (Mann-Whitney test, p < 0.0001). FGFR2 showed the highest number in the epithelium during the 7th dw (111/mm2) and 9th dw (87/mm2) compared to 6th, 8th and 16th dw (Kruskal-Wallis test, p < 0.001, p < 0.0001, p < 0.01 respectively). The highest number of FGFR2 positive cells in the mesenchyme was observed during the 9th dw (26/mm2), compared to the 6th, 7th,8th and 16th dw (Kruskal-Wallis test, p < 0.0001), while the number of FGFR2 positive cells in the epithelium was significantly higher than in the mesenchyme (Mann-Whitney test, p < 0.0001). CTGF was negative in both epithelium and mesenchyme during all except the 16th dw in the mesenchyme where it co-localized with FGFR2. FGFR1 and FGFR2 might be essential for epithelial-mesenchymal interactions that determine epithelial branching and mesenchymal growth during early lung development. Sudden increase in FGF1 in the epithelium and FGF2 in the mesenchyme in the foetus at 9th dw could be associated with the onset of foetal breathing movements. CTGF first appear during the foetal lung development.

Early Response of CD8+ T Cells in COVID-19 Patients.

Healthy and controlled immune response in COVID-19 is crucial for mild forms of the disease. Although CD8+ T cells play important role in this response, there is still a lack of studies showing the gene expression profiles in those cells at the beginning of the disease as potential predictors of more severe forms after the first week. We investigated a proportion of different subpopulations of CD8+ T cells and their gene expression patterns for cytotoxic proteins (perforin-1 (PRF1), granulysin (GNLY), granzyme B (GZMB), granzyme A (GZMA), granzyme K (GZMK)), cytokine interferon-γ (IFN-γ), and apoptotic protein Fas ligand (FASL) in CD8+ T cells from peripheral blood in first weeks of SARS-CoV-2 infection. Sixteen COVID-19 patients and nine healthy controls were included. The absolute counts of total lymphocytes (p = 0.007), CD3+ (p = 0.05), and CD8+ T cells (p = 0.01) in COVID-19 patients were significantly decreased compared to healthy controls. In COVID-19 patients in CD8+ T cell compartment, we observed lower frequency effector memory 1 (EM1) (p = 0.06) and effector memory 4 (EM4) (p < 0.001) CD8+ T cells. Higher mRNA expression of PRF1 (p = 0.05) and lower mRNA expression of FASL (p = 0.05) at the fifth day of the disease were found in COVID-19 patients compared to healthy controls. mRNA expression of PRF1 (p < 0.001) and IFN-γ (p < 0.001) was significantly downregulated in the first week of disease in COVID-19 patients who progressed to moderate and severe forms after the first week, compared to patients with mild symptoms during the entire disease course. GZMK (p < 0.01) and FASL (p < 0.01) mRNA expression was downregulated in all COVID-19 patients compared to healthy controls. Our results can lead to a better understanding of the inappropriate immune response of CD8+ T cells in SARS-CoV2 with the faster progression of the disease.

Decreased Expression of Cytotoxic Proteins in Decidual CD8+ T Cells in Preeclampsia.

In our study, we aimed to establish expression of cytotoxic CD8+ T cells in the decidua basalis and the maternal peripheral blood (mPBL) of severe and mild preeclampsia (PE) and compare to healthy pregnancies. Decidual tissue and mPBL of 10 women with mild PE, 10 women with severe PE, and 20 age-matched healthy pregnancy controls were analyzed by double immunofluorescence and qPCR, respectively. By double immunofluorescence staining, we found a decreased total number of cells/mm2 in decidua basalis of granulysin (GNLY)+ (p ˂ 0.0001), granzyme B (GzB)+(p ˂ 0.0001), GzB+CD8+(p ˂ 0.0001), perforin (PRF1)+ (p ˂ 0.0001), and PRF1+CD8+ (p ˂ 0.01) in the severe PE compared to control group. Additionally, we noticed the trend of lower mRNA expression for GNLY, granzyme A (GZMA), GzB, and PRF1 in CD8+ T cells of mPBL in mild and severe PE, with the latter marker statistically decreased in severe PE (p ˂ 0.001). Forkhead box P3 (FOXP3) mRNA in CD8+ T cells mPBL was increased in mild PE (p ˂ 0.001) compared to controls. In conclusion, severe PE is characterized by altered expression of cytotoxic CD8+ T cells in decidua and mPBL, suggesting their role in pathophysiology of PE and fetal-maternal immune tolerance.