The Incidence of Necrotizing Enterocolitis and Late-Onset Sepsis during the COVID-19 Pandemic in Sweden: A Population-Based Cohort Study.

INTRODUCTION: The effect of the pandemic restrictions in the NICUs is not well studied. Necrotizing enterocolitis (NEC) is characterized by intestinal inflammation and bacterial invasion. This study aimed to investigate whether the incidence of NEC has changed during the COVID-19 pandemic in Sweden and whether it was associated with a change in the frequency of extremely preterm births. METHODS: Data were retrieved from the Swedish Neonatal Quality Register (SNQ) for infants registered between January 2017 and December 2021 born below a gestational age of 35 weeks. The registry completeness is 98-99%. The diagnosis of NEC was the primary outcome. Generalized linear model analysis was used to calculate the risk ratio for NEC. RESULTS: Totally 13,239 infants were included. 235 (1.8%) infants developed NEC, out of which 91 required surgical treatment. 8,967 infants were born before COVID-19 pandemic and 4,272 during. Median gestational age at birth was 32.8 weeks in both periods. The incidence of NEC was significantly lower during COVID-19 pandemic compared to the prior period (1.43 vs. 1.94%, p 0.037), but not the incidence of surgical NEC. The crude risk ratio of developing NEC during COVID-19 pandemic was 0.74 (95% CI: 0.55-0.98). The incidence of late-onset sepsis with positive culture was also declined during COVID-19 (3.21 vs. 4.15%, p value 0.008). CONCLUSION: While we found significant reduction in the incidence of NEC and culture-positive late-onset sepsis during the COVID-19 pandemic, the number of extremely preterm births was unchanged.

Collagen type IV alpha 1 chain (COL4A1) expression in the developing human lung.

BACKGROUND: Collagen type IV alpha 1 chain (COL4A1) in the basement membrane is an important component during lung development, as suggested from animal models where COL4A1 has been shown to regulate alveolarization and angiogenesis. Less is known about its role in human lung development. Our aim was to study COL4A1 expression in preterm infants with different lung maturational and clinical features. METHODS: COL4A1 expression in 115 lung samples from newborn infants (21-41 weeks' gestational age; 0-228 days' postnatal age [PNA]) was studied by immunohistochemistry combined with digital image analysis. Cluster analysis was performed to find subgroups according to immunohistologic and clinical data. RESULTS: Patients were automatically categorized into 4 Groups depending on their COL4A1 expression. Expression of COL4A1 was mainly extracellular in Group 1, low in Group 2, intracellular in Group 3, and both extra- and intracellular in Group 4. Intracellular/extracellular ratio of COL4A1 expression related to PNA showed a distinctive postnatal maturational pattern on days 1-7, where intracellular expression of COL4A1 was overrepresented in extremely preterm infants. CONCLUSIONS: COL4A1 expression seems to be highly dynamic during the postnatal life due to a possible rapid remodeling of the basement membrane. Intracellular accumulation of COL4A1 in the lungs of extremely premature infants occurs more frequently between 1 and 7 postnatal days than during the first 24 hours. In view of the lung arrest described in extremely preterm infants, the pathological and/or developmental role of postnatally increased intracellular COL4A1 as marker for basement membrane turnover, needs to be further investigated.

Paneth cell proteins DEFA6 and GUCA2A as tissue markers in necrotizing enterocolitis.

Previous studies suggest that Paneth cells are involved in NEC development. Defensin alpha 6 (DEFA6) and guanylate cyclase activator 2A (GUCA2A) are selective protein markers of Paneth cells. The objective was to explore DEFA6 and GUCA2A expression in intestinal tissue samples from newborn infants with and without NEC. Tissue samples from histologically intact intestine were analyzed from 70 infants: 43 underwent bowel resection due to NEC and 27 controls were operated due to conditions such as intestinal atresia, dysmotility, aganglionosis, pseudo-obstruction or volvulus. Each tissue sample was immunohistochemically stained for DEFA6 and GUCA2A. Semi-automated digital image analysis was performed to determine protein expression. Clinical data and protein expressions were compared between the groups. DEFA6 expression was lower in the NEC group (p = 0.006). Low DEFA6 correlated with risk of developing NEC in a logistic regression analysis, independently of gestational age and birth weight (OR 0.843 [CI 0.732-0.971]; p = 0.018). GUCA2A expression did not differ between the two groups. CONCLUSION: Lower expression of DEFA6 together with intact GUCA2A expression indicates that NEC patients have well-defined Paneth cells but diminished defensin activity. Our results suggest that DEFA6 could be used as a biomarker for NEC. WHAT IS KNOWN: • Previous studies of defensin activity in NEC have been inconsistent, showing that defensin levels may be increased or diminished in NEC. GUCA2A has to our knowledge never been studied in NEC. WHAT IS NEW: • This study benchmarks two specific Paneth cell markers (DEFA6 and GUCA2A) and their activity in individuals with and without NEC. • The key finding is that the NEC group had a lower DEFA6 expression compared to the Controls, while the expression of GUCA2A did not differ between the groups.

Vascular adhesion protein-1 expression is reduced in the intestines of infants with necrotizing enterocolitis: an observational research study.

BACKGROUND: Necrotizing enterocolitis (NEC) is an inflammatory bowel disease in preterm neonates with high morbidity and mortality. The only treatment available is supportive with broad-spectrum antibiotics and gastrointestinal rest. Better understanding of the pathogenesis is crucial for the development of new therapies. Vascular adhesion protein-1 (VAP-1), expressed in human blood vessels and lymphatic, plays a crucial role in the pathogenesis of inflammatory diseases in adults. The aim of the study was to investigate the VAP-1 expression in the intestines of infants affected by NEC. METHODS: Intestinal tissues from 42 preterm infants with NEC were examined with immunohistochemical staining using antibodies against VAP-1 and semi-automated digital image analysis was performed to determine tissue protein expression of VAP-1 in blood vessels located in the submucosa. Intestinal tissue from 26 neonates that underwent laparotomy and ileostomy due to other intestinal surgical conditions served as controls. Clinical data and protein expression were compared between the NEC-group and Controls. RESULTS: Mean gestational age was lower in NEC infants compared to controls, 26.6 ± 3.0 gestational weeks versus 36.5 ± 4.0 (p < 0.001) but without any significant difference in median postnatal age at surgery; for NEC 8 (5-27) days and for controls 3 (1-36) days (p = 0.6). Low VAP-1 correlated with increased risk for developing NEC in the logistic regression (p < 0.001). Multiple linear regression showed that both gestational age and NEC were independent predictors of VAP-1 expression. CONCLUSION: VAP-1 may play a role in the pathogenesis of NEC. Diminished expression of VAP-1 independent of maturation could indicate arrested vascular development in infants suffering from NEC. Further studies are needed to elucidate the role of VAP-1 in NEC.

Early Postnatal Comprehensive Biomarkers Cannot Identify Extremely Preterm Infants at Risk of Developing Necrotizing Enterocolitis.

Background: Necrotizing enterocolitis (NEC) is a fatal disease where current diagnostic tools are insufficient for preventing NEC. Early predictive biomarkers could be beneficial in identifying infants at high risk of developing NEC. Objective: To explore early biomarkers for predicting NEC in extremely preterm infants (EPIs). Methods: Blood samples were collected on day 2 (median 1.7; range 1.5-2.0) from 40 EPI (median 25 gestational weeks; range 22-27): 11 developed NEC and 29 did not (controls). In each infant, 189 inflammatory, oncological, and vascular proteomic biomarkers were quantified through Proximity Extension Assay. Biomarker expression and clinical data were compared between the NEC group and Controls. Based on biomarker differences, controls were sorted automatically into three subgroups (1, 2, and 3) by a two-dimensional hierarchical clustering analysis. Results: None of the biomarkers differed in expression between all controls and the NEC group. Two biomarkers were higher in Control 1, and 16 biomarkers were lower in Control group 2 compared with the NEC group. No biomarker distinguished Control 3 from the NEC group. Perinatal data were similar in the whole population. Conclusions: Early postnatal comprehensive biomarkers do not identify EPIs at risk of developing NEC in our study. Future studies of predictors of NEC should include sequential analysis of comprehensive proteomic markers in large cohorts.

The value of autopsy in preterm infants at a Swedish tertiary neonatal intensive care unit 2002-2018.

Reliable data on causes of death (COD) in preterm infants are needed to assess perinatal care and current clinical guidelines. In this retrospective observational analysis of all deceased preterm infants born < 37 weeks' gestational age (n = 278) at a Swedish tertiary neonatal intensive care unit, we compared preliminary COD from Medical Death Certificates with autopsy defined COD (2002-2018), and assessed changes in COD between two periods (period 1:2002-2009 vs. period 2:2011-2018; 2010 excluded due to centralized care and seasonal variation in COD). Autopsy was performed in 73% of all cases and was more than twice as high compared to national infant autopsy rates (33%). Autopsy revised or confirmed a suspected preliminary COD in 34.9% of the cases (23.6% and 11.3%, respectively). Necrotizing enterocolitis (NEC) as COD increased between Period 1 and 2 (5% vs. 26%). The autopsy rate did not change between the two study periods (75% vs. 71%). We conclude that autopsy determined the final COD in a third of cases, while the incidence of NEC as COD increased markedly during the study period. Since there is a high risk to determine COD incorrectly based on clinical findings in preterm infants, autopsy remains a valuable method to obtain reliable COD.

CD44 and RHAMM expression patterns in the human developing lung.

BACKGROUND: The hyaluronan (HA) receptors CD44 and RHAMM (CD168) are involved in cellular proliferation, differentiation, and motility. As previously investigated, HA and RHAMM expression in human neonatal lungs correlates to gestational age (GA) and air content. METHODS: CD44 immunofluorescence was analyzed in postmortem lung samples from infants (n = 93; 22-41 GA) by digital image analysis together with clinical data, including RHAMM expression, lung air, and HA content by hierarchical clustering. RESULTS: Five groups were defined according to RHAMM/CD44 expression, GA, and postnatal age (PNA): extremely to very preterm (EVP; 22-31 GA; Groups 1-2), moderately preterm to term (MPT; 31-41 GA; Groups 3-4), and mixed preterm to term (27-40 GA; Group 5). CD44 correlated linearly with RHAMM in MPT (r = 0.600; p < 0.004). In EVP, high CD44 and low RHAMM corresponded with high PNA and lung air content independently of HA and GA (Group 1 vs 2; p < 0.05). In MPT, high and low CD44 corresponded with low and high RHAMM independently of GA, HA, and lung air content (Group 3 vs 4; p < 0.001). No correlation between CD44 and GA/PNA at death was observed. CONCLUSIONS: A linear correlation between CD44 and RHAMM expression occurs during the late saccular phase of lung development at birth, whereas postnatal influences on CD44 and RHAMM expression in extremely to very preterm infants cannot be excluded. IMPACT: The interplay between CD44 and RHAMM, two receptors of hyaluronic acid, can be dependent on the lung developmental stage at birth. This is the second study that analyzes the distribution pattern of CD44 in the human lung during development and the first study performed with quantitative analysis of CD44 expression together with RHAMM expression in the human lung. Our results suggest a relationship in a subset of infants between CD44 and RHAMM expression, which appears at birth during the late saccular stage but not during the earlier stages of lung development.

Cluster Analysis of Early Postnatal Biochemical Markers May Predict Development of Retinopathy of Prematurity.

Purpose: Growth factors and inflammatory and angiogenetic proteins are involved in the development of retinopathy of prematurity (ROP). However, no early biochemical markers are in clinical use to predict ROP. By performing cluster analysis of multiple biomarkers, we aimed to determine patient groups with high and low risk for developing ROP. Methods: In total, 202 protein markers in plasma were quantified by proximity extension assay from 35 extremely preterm infants on day 2 of life. Infants were sorted in groups by automated two-dimensional hierarchical clustering of all biomarkers. ROP was classified as stages I to III with or without surgical treatment. Predictive biomarkers were evaluated by analysis of variance and detected differences by two-sided paired t-test with Bonferroni corrections for multiple comparisons. Results: Differences in 39 biochemical markers divided infants without ROP into two control groups (control 1, n = 7; control 2, n = 5; P < 0.05). Sixty-six biochemical markers defined differences between the control groups (n = 13) and all ROP infants (n = 23; P < 0.05). PARK7, VIM, MPO, CD69, and NEMO were markedly increased in control 1 compared to all ROP infants (P < 0.001). Lower TNFRSF4 and higher HER2 and GAL appeared in infants with ROP as compared to control 1 and/or 2 (P < 0.05, respectively). Conclusions: Our data suggest that early elevated levels of PARK7, VIM, MPO, CD69, and NEMO may be associated with lower risk of developing ROP. Lower levels of TNFRSF4 with higher levels of HER2 and GAL may predict ROP development. Translational Relevance: Cluster analysis of early postnatal biomarkers may help to identify infants with low or high risk of developing ROP.

Diminished DEFA6 Expression in Paneth Cells Is Associated with Necrotizing Enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is the most common gastrointestinal disorder in premature infants with a high morbidity and mortality. Paneth cell dysfunction has been suggested to be involved in the pathogenesis of NEC. Defensin alpha-6 (DEFA6) is a specific marker for Paneth cells acting as part of the innate immunity in the human intestines. The aim of this study was to investigate the expression of DEFA6 in infants with NEC. MATERIALS AND METHODS: Infants who underwent bowel resection for NEC at level III NICU in Sweden between August 2004 and September 2013 were eligible for the study. Macroscopically vital tissues were selected for histopathological evaluation. All infants in the control group underwent laparotomy and had ileostomy due to dysmotility, and samples were taken from the site of the stoma. DEFA6 expression was studied by immunohistochemistry. Digital image analysis was used for an objective and precise description of the samples. RESULTS: A total of 12 infants were included in the study, eight with NEC and four controls. The tissue samples were taken from the colon (n = 1), jejunum (n = 1), and ileum (n = 10). Both the NEC and control groups consisted of extremely premature and term infants (control group: 25-40 gestational weeks, NEC group: 23-39 gestational weeks). The postnatal age at the time of surgery varied in both groups (control group: 4-47 days, NEC group: 4-50 days). DEFA6 expression in the NEC group was significantly lower than that in the control group and did not correlate with gestational age. CONCLUSION: The diminished DEFA6 expression in Paneth cells associated with NEC in this study supports the hypothesis that alpha-defensins are involved in the pathophysiology of NEC. Future studies are needed to elucidate the role of alpha-defensins in NEC aiming at finding preventive and therapeutic strategies against NEC.

The receptor for hyaluronan-mediated motility (RHAMM) expression in neonatal bronchiolar epithelium correlates negatively with lung air content.

INTRODUCTION: Hyaluronan (HA) and the receptor for hyaluronan-mediated motility (RHAMM) may play an important role in lung development. We examined the expression of HA content and RHAMM during postnatal lung development by analyzing human lung specimens from newborn infants with a variety of lung diseases at different gestational (GA) and postnatal (PNA) ages. MATERIALS AND METHODS: Ninety-four patients were evaluated. Immunohistochemical RHAMM expression was studied with digital image analysis, followed by hierarchical cluster analysis of both these data and clinical data to define subgroups. The air content of the lung was determined by computerized analysis. HA content was estimated by radiometric assay. RESULTS: Cluster analysis defined six distinct patient groups (Group 1-2: 34-41 weeks GA; Group 3-5: 23-27 weeks GA; Group 6: mixed population). Group 1-5 showed individual patterns in RHAMM expression and HA content (Group 1: high RHAMM/low HA; Group 2: low RHAMM/low HA; Group 3: low RHAMM/low HA; Group 4: low RHAMM/high HA; Group 5: high RHAMM/high HA). HA content decreased with increasing PNA independently of GA. Negative correlation was observed between air content and RHAMM expression in the bronchiolar epithelium irrespective of clustered groups. Lung hypoplasia appeared in two distinctive groups, with significant differences in lung development and RHAMM expression. CONCLUSIONS: RHAMM expression may show dynamic changes during pathological processes in the neonatal lung. The distribution of RHAMM in the lung tissue is heterogeneous with a predominance to the bronchiolar epithelium. We found a negative correlation between lung air content and RHAMM expression in bronchiolar epithelium.

Inhibition of protein phosphatase-1 and -2A decreases the chemosensitivity of leukemic cells to chemotherapeutic drugs.

The phosphorylation of key proteins balanced by protein kinases and phosphatases are implicated in the regulation of cell cycle and apoptosis of malignant cells and influences anticancer drug actions. The efficacy of daunorubicin (DNR) in suppression of leukemic cell survival was investigated in the presence of tautomycin (TM) and calyculin A (CLA), specific membrane permeable inhibitors of protein phosphatase-1 (PP1) and -2A (PP2A), respectively. CLA (50 nM) or TM (1µM) suppressed viability of THP-1 and KG-1 myeloid leukemia cell lines to moderate extents; however, they significantly increased survival upon DNR-induced cell death. CLA increased the phosphorylation level of Erk1/2 and PKB/Akt kinases, the retinoblastoma protein (pRb), decreased caspase-3 activation by DNR and increased the phosphorylation level of the inhibitory sites (Thr696 and Thr853) in the myosin phosphatase (MP) target subunit (MYPT1) as well as in a 25kDa kinase-enhanced phosphatase inhibitor (KEPI)-like protein. TM induced enhanced phosphorylation of pRb only, suggesting that this event may be a common factor upon CLA-induced PP2A and TM-induced PP1 inhibitory influences on cell survival. Silencing PP1 by siRNA in HeLa cells, or overexpression of Flag-KEPI in MCF-7 cells coupled with inducing its phosphorylation by PMA or CLA, resulted in increased phosphorylation of pRb. Our results indicate that PP1 directly dephosphorylates pRb, while PP2A might have an indirect influence via mediating the phosphorylation level of PP1 inhibitory proteins. These data imply the importance of PP1 inhibitory proteins in controlling the phosphorylation state of key proteins and regulating drug sensitivity and apoptosis in leukemic cells.

Drug sensitivity patterns of HHV8 carrying body cavity lymphoma cell lines.

BACKGROUND: Primary effusion lymphoma (PEL) is a rare KSHV/HHV8-associated high-grade non-Hodgkin's lymphoma (NHL) of B-cell origin, characterized by serous effusions in body cavities. Most patients are HIV-infected men with severe immunosuppression and other HHV8-associated diseases such as Kaposi's sarcoma (KS). The prognosis for those infected is poor, with a median survival of less than 6 months in most cohorts. Sustained complete remission is rare. High-dose chemotherapy regimens are used to improve remission rate and survival. The aim of the present study was to compare the drug sensitivity pattern of the available primary effusion (body cavity based) lymphoma-derived cell lines in order to find additional, potentially effective drugs that are not included in current chemotherapy treatment protocols. METHODS: We have analyzed 11 cell lines against 27 frequently used cytostatic drugs in short term (3 days) survival assays using automated high throughput confocal microscopy. RESULTS: All cell lines showed a distinct, individual drug sensitivity pattern. Considering the in vitro used and clinically achieved drug concentration, Vinorelbine, Paclitaxel, Epirubicin and Daunorubicin were the most effective drugs. CONCLUSIONS: We suggest that inclusion of the above drugs into PEL chemotherapy protocols may be justified. The heterogeneity in the drug response pattern however indicated that assay-guided individualized therapy might be required to optimize therapeutic response.

High-throughput live-cell imaging reveals differential inhibition of tumor cell proliferation by human fibroblasts.

Increasing evidence indicates that cancer development requires changes both in the precancerous cells and in their microenvironment. To study one aspect of the microenvironmental control, we departed from Michael Stoker's observation (Stroker et al, J Cell Sci 1966;1:297-310) that normal fibroblasts can inhibit the growth of admixed cancer cells (neighbour suppression). We have developed a high-throughput microscopy and image analysis system permitting the examination of live mixed cell cultures growing on 384-well plates, at the single cell level and over time. We have tested the effect of 107 samples of low passage number (<5) primary human fibroblasts from pediatric and adult donors, on the growth of six human tumor cell lines. Three of the lines were derived from prostate carcinomas, two from lung carcinomas and one was an EBV transformed lymphoblastoid line. Labeled tumor cells were grown in the presence of unlabeled fibroblasts. The majority of the tested fibroblasts inhibited the proliferation of the tumor cells, compared to the control cultures where labeled tumor cells were co-cultured with unlabeled tumor cells. The proliferation inhibiting effect of the fibroblasts differed depending on their site of origin and the age of the donor. Inhibition required direct cell contact. Mouse 3T3 fibroblasts inhibited the growth of SV40-transformed 3T3 cells and human tumor cells, showing that the inhibitory effect could prevail across the species barrier. Our high-throughput system allows the quantitative analysis of the inhibitory effect of fibroblasts on the population level and the exploration of differences depending on the source of the normal cells.

Chronic lymphoid leukemia cells are highly sensitive to the combination of prednisolone and daunorubicin, but much less to doxorubicin or epirubicin.

OBJECTIVE: To generate a comprehensive map of the drug sensitivity of chronic lymphoid leukemia cells (CLL) using a newly developed in vitro drug-sensitivity assay based on automated evaluation of cell viability on single-cell level. MATERIALS AND METHODS: Primary CLL cells from 77 patients were tested using automated digital fluorescence microscopy. The effect of 27 frequently used chemotherapeutic agents was measured in short-term fluorescence survival assay. To avoid typical in vitro artifacts such as growth factor depletion and oxidative damage, the cell were cultured in a novel, total human blood lysate-based medium (OmniSanguine) in order to preserve the composition of growth factor flora and redox conditions of the in vivo environment. RESULTS: CLL cells from different patients showed considerable heterogeneity in their drug-sensitivity patterns. This pattern was stable even after in vitro activation of cell proliferation. Half of the samples were sensitive to fludarabine and chlorambucil. Daunorubicin was the most potent drug. It was effective in 75 of 77 cases. In addition, daunorubicin and prednisolone showed a strong synergistic effect. CONCLUSIONS: We suggest that the combination of low-dose daunorubicin and prednisolone might be an additional treatment option for therapy-resistant cases of CLL.

NK cell-mediated lysis is essential to kill Epstein-Barr virus transformed lymphoblastoid B cells when using rituximab.

Rituximab is a humanized chimeric monoclonal antibody, targeted against the pan B cell marker CD20. It is frequently used to treat a variety of B cell lymphomas and immunosuppression associated lymphoproliferations such as posttransplant lymphoproliferative disorder (PTLD). The response rate of rituximab treatment is 65%, but the exact in vivo mechanism of action is not yet fully understood, although antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct induction of apoptosis have been suggested as effector mechanism. Rituximab may affect different types of lymphomas through different mechanisms. As lymphoblastoid cell lines (LCLs) are well-established in vitro models of PTLD, we investigated the effect of rituximab on these cells using a custom built automated laser confocal fluorescent microscope. We found that rituximab alone was not effective at inducing cell death of EBV-transformed B cells. The antibody was effective in the complement-mediated CDC. Rituximab could induce NK cell-mediated ADCC but it was more effective in the presence of untreated fresh human plasma compared to heat-inactivated human plasma. Our data suggest that complement-enhanced NK-mediated ADCC is required for effective rituximab mediated killing of EBV-transformed B cells. Determining and monitoring of serum complement levels and in vitro killing efficacy of NK cells of PTLD patients might help to predict resistant cases to rituximab therapy. On the other hand our results suggest a possibility that rituximab should be combined only with cytotoxic drugs that spare NK function when treating PTLD patients.

Myosin phosphatase interacts with and dephosphorylates the retinoblastoma protein in THP-1 leukemic cells: its inhibition is involved in the attenuation of daunorubicin-induced cell death by calyculin-A.

Reversible phosphorylation of the retinoblastoma protein (pRb) is an important regulatory mechanism in cell cycle progression. The role of protein phosphatases is less understood in this process, especially concerning the regulatory/targeting subunits involved. It is shown that pretreatment of THP-1 leukemic cells with calyculin-A (CL-A), a cell-permeable phosphatase inhibitor, attenuated daunorubicin (DNR)-induced cell death and resulted in increased pRb phosphorylation and protection against proteolytic degradation. Protein phosphatase-1 catalytic subunits (PP1c) dephosphorylated the phosphorylated C-terminal fragment of pRb (pRb-C) slightly, whereas when PP1c was complexed to myosin phosphatase target subunit-1 (MYPT1) in myosin phosphatase (MP) holoenzyme dephosphorylation was stimulated. The pRb-C phosphatase activity of MP was partially inhibited by anti-MYPT1(1-296) implicating MYPT1 in targeting PP1c to pRb. MYPT1 became phosphorylated on both inhibitory sites (Thr695 and Thr850) upon CL-A treatment of THP-1 cells resulting in the inhibition of MP activity. MYPT1 and pRb coprecipitated from cell lysates by immunoprecipitation with either anti-MYPT1 or anti-pRb antibodies implying that pRb-MYPT1 interaction occurred at cellular levels. Surface plasmon resonance-based experiments confirmed binding of pRb-C to both PP1c and MYPT1. In control and DNR-treated cells, MYPT1 and pRb were predominantly localized in the nucleus exhibiting partial colocalization as revealed by immunofluorescence using confocal microscopy. Upon CL-A treatment, nucleo-cytoplasmic shuttling of both MYPT1 and pRb, but not PP1c, was observed. The above data imply that MP, with the targeting role of MYPT1, may regulate the phosphorylation level of pRb, thereby it may be involved in the control of cell cycle progression and in the mediation of chemoresistance of leukemic cells.

Granulocyte colony stimulating factor increases drug resistance of leukaemic blast cells to daunorubicin.

Acute leukaemia is known as the most common cancer in childhood. Febrile neutropenia is a common serious side effect of the cytostatic treatment of malignancies. The clinical use of Granulocyte Colony Stimulating Factor (G-CSF) has become widespread to minimize chemotherapy-induced myelosuppression and febrile neutropenia in childhood solid tumors, acute lymphoid leukaemia (ALL) and in several trials with AML. In case of ALL this seems to be reasonable because, due to the absence of G-CSF receptor (G-CSFR) on the surface of normal lymphoid cells, G-CSF does not have any influence on the pathways of proliferation and differentiation of lymphoid lineage cells. It has been suggested, however, that ALL blasts with B or T cell surface antigens as well as biphenotypic leukaemia cells express G-CSFR, and they are able to respond to exogenously added G-CSF with proliferation. In this study we investigated how G-CSF might influence the sensitivity of leukemic cells to daunorubicin induced cell death using MTT assay, flow cytometry and Western blot analysis. After pretreatment of KG-1 leukaemic cells with G-CSF a moderate increase in the resistance of these cells to daunorubicin could be observed. These results draw attention to the risk of G-CSF application as an adjuvant therapy of childhood ALL. In addition, adjuvant treatment of AML patients with G-CSF in order to prevent neutropenia, or its use in priming regimens might result resistance to daunorubicin.

Effect of frequently used chemotherapeutic drugs on cytotoxic activity of human cytotoxic T-lymphocytes.

Tumors are considered to be possible targets of immunotherapy using stimulated and expanded cytotoxic T-lymphocytes (CTL). It is important to consider the drug-induced effects when chemotherapeutic regimens and CTL-mediated immunotherapy is planned to be used in parallel. In this study, we characterized the effect of 29 frequently used chemotherapeutic agents on the cytotoxic activity of autologous and allogeneic CTLs. We found that treatment of CTLs with the following drugs: docetaxel, vincristine, chlorambucil, mitomycin C, oxaliplatin, doxorubicin, and bleomycin effectively inhibited CTL-mediated killing, without affecting their viability. On the other hand, the following drugs enhanced or permitted efficient CTL-mediated killing in vitro at concentrations comparable with the maximally achieved therapeutic concentration in vivo in humans: daunorubicin, prednisolone, vinorelbine, cisplatin, methotrexate, hydroxyurea, cytarabine, cyclophosphamide, topotecan, epirubicin, fluorouracil, carboplatin, asparaginase, 6-mercaptopurine, and bortezomib. Our results could potentially be used in the future to design new CTL-based adjuvant immunotherapy protocols.

Hodgkin-lymphoma-derived cells show high sensitivity to dactinomycin and paclitaxel.

Depending on stage and risk factors, up to 30% of patients with advanced Hodgkin lymphoma (HL) progress or relapse. Patients with pleural effusions have a particularly poor prognosis and this stage of HL is regularly resistant to chemotherapy. All currently available HL cell lines are derived from late stage HL patients. In the present study we measured the sensitivity of these HL lines against the 26 most frequently used cytostatic drugs. We used a novel fluorescent short-term survival assay where the cell was incubated with the drugs for 4 days. The precise number of differentially stained live and dead cells was determined using a custom-built automated laser confocal fluorescent microscope. We found that HL cells, independently of their origin, showed very similar sensitivity patterns for several of the drugs. All HL cell lines were highly sensitive to dactinomycin, paclitaxel and etoposide. Our data suggest that the inclusion of dactinomycin and paclitaxel into chemotherapy protocols against late stage Hodgkin lymphoma with pleural effusion may be justified.

HHV-8 encoded LANA-1 alters the higher organization of the cell nucleus.

The latency-associated nuclear antigen (LANA-1) of Human Herpes Virus 8 (HHV-8), alternatively called Kaposi Sarcoma Herpes Virus (KSHV) is constitutively expressed in all HHV-8 infected cells. LANA-1 accumulates in well-defined foci that co-localize with the viral episomes. We have previously shown that these foci are tightly associated with the borders of heterochromatin 1. We have also shown that exogenously expressed LANA-1 causes an extensive re-organization of Hoechst 33248 DNA staining patterns of the nuclei in non-HHV-8 infected cells 2. Here we show that this effect includes the release of the bulk of DNA from heterochromatic areas, in both human and mouse cells, without affecting the overall levels of heterochromatin associated histone H3 lysine 9 tri-methylation (3MK9H3). The release of DNA from the heterochromatic chromocenters in LANA-1 transfected mouse cells co-incides with the dispersion of the chromocenter associated methylcytosin binding protein 2 (MECP2). The localization of 3MK9H3 to the remnants of the chromocenters remains unaltered. Moreover, exogeneously expressed LANA-1 leads to the relocation of the chromocenters to the nuclear periphery, indicating extensive changes in the positioning of the chromosomal domains in the LANA-1 harboring interphase nucleus. Using a series of deletion mutants we have shown that the chromatin rearranging effects of LANA-1 require the presence of a short (57 amino acid) region that is located immediately upstream of the internal acidic repeats. This sequence lies within the previously mapped binding site to histone methyltransferase SUV39H1. We suggest that the highly concentrated LANA-1, anchored to the host genome in the nuclear foci of latently infected cells and replicated through each cell generation, may function as "epigenetic modifier". The induction of histone modification in adjacent host genes may lead to altered gene expression, thereby contributing to the viral oncogenesis.