2-Amino-Pyrrole-Carboxylate Attenuates Homology-Mediated DNA Repair and Sensitizes Cancer Cells to Doxorubicin.

Despite a high efficacy of chemotherapy in cancer treatment, acquired resistance of tumors to certain chemotherapeutic agents and frequent side effects remain the major factors of unfavorable prognosis in most cancer patients with unresectable, metastatic and recurrent forms of the disease. The discovery of novel molecular targets in tumors and development of new therapeutic approaches to enhance the efficiency of chemotherapeutic agents remain the biggest challenges in current oncology. Here we examined the ability of pyrrole-based heterocyclic compound 2-APC to sensitize tumor cells to the topoisomerase II inhibitor doxorubicin. The study was performed on human cancer cell lines treated with 2-APC, paclitaxel, and doxorubicin. Expression of DNA repair was investigated by Western blotting, whereas protein-protein interactions were examined by co-immunoprecipitation. The synergism between the chemotherapeutic agents was assessed with the Synergy Finder program. Doxorubicin exhibited moderate cytotoxic effect against cancer cell lines (in particular, osteosarcoma cell lines). 2-APC in non-toxic concentrations substantially potentiated the cytotoxic effect of doxorubicin and induced apoptosis of cancer cells. This activity of 2-APC was due to its ability to impair DNA damage repair by decreasing the content of Rad51 recombinase via promoting its proteasomal degradation. Similar effects were observed for paclitaxel, which affects tubulin polymerization. Therefore, chemotherapeutic agents and chemical compounds interfering with the microtubule dynamics can potentiate the cytotoxic effects of DNA-damaging chemotherapeutic agents via impairment of DNA damage repair mechanisms in cancer cells.

Development of quantitative assay for simultaneous measurement of purine metabolites and creatinine in biobanked urine by liquid chromatography-tandem mass spectrometry.

Purine metabolism is essential for all known living creatures, including humans in whom elevated serum concentration of purine break-down product uric acid (UA) is probably an independent risk factor for mortality, type 2 diabetes and cardiovascular events. An automated multiplex assay that measures several purine metabolites could therefore prove useful in many areas of medical, veterinary and biological research. The aim of the present work was to develop a sensitive LC-MS/MS method for simultaneous quantitation of xanthine, hypoxanthine, UA, allantoin, and creatinine in biobanked urine samples. This article describes details and performance of the new method studied in 55 samples of human urine. Archival sample preparation and effect of storage conditions on stability of the analytes are addressed. The intra-day and inter-day coefficients of variation were small for all the analytes, not exceeding 1% and 10%, respectively. Measurements of UA and creatinine in biobanked urine showed good agreement with values obtained using routine enzymatic assays on fresh urine. Spearman's correlation coefficients were 0.869 (p < .001) for creatinine and 0.964 (p < .001) for UA. Conclusion: the newly developed LC-MS/MS method allows reliable quantitative assessment of xanthine, hypoxanthine, allantoin, UA and creatinine. The proposed pre-analytical processing makes the method suitable for both fresh and biobanked urine stored frozen at -80 °C for at least 5.5 years.

Computational-Based Discovery of the Anti-Cancer Activities of Pyrrole-Based Compounds Targeting the Colchicine-Binding Site of Tubulin.

Despite the tubulin-binding agents (TBAs) that are widely used in the clinic for cancer therapy, tumor resistance to TBAs (both inherited and acquired) significantly impairs their effectiveness, thereby decreasing overall survival (OS) and progression-free survival (PFS) rates, especially for the patients with metastatic, recurrent, and unresectable forms of the disease. Therefore, the development of novel effective drugs interfering with the microtubules' dynamic state remains a big challenge in current oncology. We report here about the novel ethyl 2-amino-1-(furan-2-carboxamido)-5-(2-aryl/tert-butyl-2-oxoethylidene)-4-oxo-4,5-dihydro-1H-pyrrole-3-carboxylates (EAPCs) exhibiting potent anti-cancer activities against the breast and lung cancer cell lines in vitro. This was due to their ability to inhibit tubulin polymerization and induce cell cycle arrest in M-phase. As an outcome, the EAPC-treated cancer cells exhibited a significant increase in apoptosis, which was evidenced by the expression of cleaved forms of PARP, caspase-3, and increased numbers of Annexin-V-positive cells. By using the in silico molecular modeling methods (e.g., induced-fit docking, binding metadynamics, and unbiased molecular dynamics), we found that EAPC-67 and -70 preferentially bind to the colchicine-binding site of tubulin. Lastly, we have shown that the EAPCs indicated above and colchicine utilizes a similar molecular mechanism to inhibit tubulin polymerization via targeting the T7 loop in the ß-chain of tubulin, thereby preventing the conformational changes in the tubulin dimers required for their polymerization. Collectively, we identified the novel and potent TBAs that bind to the colchicine-binding site and disrupt the microtubule network. As a result of these events, the compounds induced a robust cell cycle arrest in M-phase and exhibited potent pro-apoptotic activities against the epithelial cancer cell lines in vitro.

The Design, Synthesis, and Biological Activities of Pyrrole-Based Carboxamides: The Novel Tubulin Inhibitors Targeting the Colchicine-Binding Site.

Microtubule targeting agents (MTAs) that interfere with the dynamic state of the mitotic spindle are well-known and effective chemotherapeutic agents. These agents interrupt the microtubule network via polymerization or depolymerization, halting the cell cycle progression and leading to apoptosis. We report two novel pyrrole-based carboxamides (CAs) (CA-61 and -84) as the compounds exhibiting potent anti-cancer properties against a broad spectrum of epithelial cancer cell lines, including breast, lung, and prostate cancer. The anti-cancer activity of CAs is due to their ability to interfere with the microtubules network and inhibit tubulin polymerization. Molecular docking demonstrated an efficient binding between these ligands and the colchicine-binding site on the tubulin. CA-61 formed two hydrogen bond interactions with THR 179 (B) and THR 353 (B), whereas two hydrogen bonds with LYS 254 (B) and 1 with ASN 101 (A) were identified for CA-84. The binding energy for CA-84 and CA-61 was -9.910 kcal/mol and -9.390 kcal/mol. A tubulin polymerization assay revealed a strong inhibition of tubulin polymerization induced by CA-61 and -84. The immunofluorescence data revealed the disruption of the tubulin assembly in CA-treated cancer cells. As an outcome of the tubulin inhibition, these compounds halted the cell cycle progression in the G2/M phase, leading to the accumulation of the mitotic cells, and further induced apoptosis. Lastly, the in vivo study indicated that CAs significantly inhibited the HCC1806 breast cancer xenograft tumor growth in a nude mouse model. Collectively, we identified the novel CAs as potent MTAs, inhibiting tubulin polymerization via binding to the colchicine-binding site, disrupting the microtubule network, and exhibiting potent pro-apoptotic activities against the epithelial cancer cell lines both in vitro and in vivo.

2-APCAs, the Novel Microtubule Targeting Agents Active Against Distinct Cancer Cell Lines.

Microtubules are known as the most attractive molecular targets for anti-cancer drugs. However, the number of serious limitations of the microtubule targeting agents (MTAs) including poor bioavailability, adverse effects (e.g., systemic and neural toxicity), and acquired resistance after initiation of MTA-based therapy remain the driving forces to develop the novel therapeutic agents effectively targeting microtubules and exhibiting potent anti-tumor activities. Here, we report the discovery of 2-amino-pyrrole-carboxamides (2-APCAs), a novel class of MTA, which effectively inhibited the growth of the broad spectrum of cancer cell lines in vitro, including various types of breast, prostate, and non-small lung cancer (NSLC), soft tissue sarcomas (STS) (e.g., leio-, rhabdomyo-, and fibrosarcomas), osteosarcomas and gastrointestinal stromal tumors (GISTs). Importantly, 2-APCAs were also effective in cancer cell lines exhibiting resistance to certain chemotherapeutic agents, including MTAs and topoisomerase II inhibitors. The anti-proliferative effect of 2-APCAs was due to their ability to interfere with the polymerization of tubulin and thereby leading to the accumulation of tumor cells in the M-phase. As an outcome of the mitotic arrest, cancer cells underwent apoptotic cell death which was evidenced by increased expression of cleaved forms of the poly-ADP-ribose polymerase (PARP) and caspase-3 and the increased numbers of Annexin V-positive cells, as well. Among the compounds exhibiting the potent anti-cancer activities against the various cancer cell lines indicated above, 2-APCA-III was found the most active. Importantly, its cytotoxic activities correlated with its highest potency to interfere with the dynamics of tubulin polymerization and inducement of cell cycle arrest in the G2/M phase. Interestingly, the cytotoxic and tubulin polymerization activities of 2-APCAs correlated with the stability of the «tubulin-2-АРСА¼ complexes, illustrating the "tubulin-2-APCA-III" complex as the most stable. Molecular docking showed that the binding site for 2-АРСА-III is located in α tubulin by forming a hydrogen bond with Leu23. Of note, single-cell electrophoresis (Comet assay) data illustrated the low genotoxic activities of 2-APCAs when compared to certain anti-cancer chemotherapeutic agents. Taken together, our study describes the novel MTAs with potent anti-proliferative and pro-apoptotic activities, thereby illustrating them as a scaffold for the development of successful chemotherapeutic anti-cancer agent targeting microtubules.

Low adiponectin is associated with diastolic dysfunction in women: a cross-sectional study from the Tromsø Study.

BACKGROUND: Heart failure with preserved ejection fraction is closely associated with diastolic dysfunction and related to obesity and female sex. We investigated whether adiponectin, an adipocyte-secreted protein hormone with cardioprotective effects, was associated with indices of diastolic dysfunction, and whether the association was sex dependent. METHODS: We conducted a cross-sectional study on 1165 women and 896 men without diabetes. We stratified the multivariable adjusted logistic regression analyses and the fractional polynomial regression analyses according to sex, with echocardiographic markers of diastolic dysfunction as dependent variables, and adiponectin as the independent variable of interest. RESULTS: Decreased adiponectin was associated with higher odds of average tissue Doppler e' < 9 in women (odds ratio [OR] 1.17 per 1 µg/mL adiponectin decrease, 95% confidence interval [CI] 1.04-1.30), but not in men (p for interaction with sex 0.04). Women, but not men, had higher odds of E/e' ratio ≥ 8 with lower adiponectin (OR 1.12 per 1 µg/mL adiponectin decrease, 95% CI 1.02-1.24, p for interaction with sex 0.04). Adiponectin in the lower sex-specific tertile was associated with increased odds of concentric left ventricular hypertrophy in women (OR 2.44, 95% CI 1.03-5.77), but with decreased odds in men (OR 0.32, 95% CI 0.11-0.88, p for interaction with sex 0.002), and decreased odds of eccentric hypertrophy in men only (OR 0.53, 95% CI 0.33-0.88, p for interaction with sex 0.02). Adiponectin in the lower sex-specific tertile was associated with moderately enlarged left atria in women only (OR 1.43, 95% CI 1.01-2.03, p for interaction with sex 0.04). Finally, adiponectin had a non-linear relationship with left ventricular mass in women only, with exponentially increasing left ventricular mass with lower adiponectin levels (p for interaction with sex 0.01). CONCLUSIONS: Low adiponectin was associated with higher odds of indices of diastolic dysfunction in women, but lower odds of indices of diastolic dysfunction in men. Lower adiponectin was associated with increased left ventricular mass in women only.

Ethyl-2-amino-pyrrole-3-carboxylates are novel potent anticancer agents that affect tubulin polymerization, induce G2/M cell-cycle arrest, and effectively inhibit soft tissue cancer cell growth in vitro.

Microtubules are known to be one of the most attractive and validated targets in cancer therapy. However, the clinical use of drugs that affect the dynamic state of microtubules has been hindered by chemoresistance and toxicity issues. Accordingly, the development of novel agents that target microtubules is needed. Here, we report the identification of novel compounds with pirrole and carboxylate structures: ethyl-2-amino-pyrrole-3-carboxylates (EAPCs) that provide potent cytotoxic activities against multiple soft tissue cancer cell lines in vitro. Using the MTS cell proliferation assay, we assessed the activity of EAPCs on various cancer cell lines including leiomyosarcoma SK-LMS-1, rhabdomyosarcoma RD, gastrointestinal stromal tumor GIST-T1, A-673 Ewing's sarcoma, and U-2 OS osteosarcoma. We found that in the majority of cases, two EAPC compounds (EAPC-20 and EAPC-24) considerably inhibited cancer cell proliferation in vitro. The growth-inhibitory effects of EAPC-20 and EAPC-24 were time and dose dependent. The molecular mechanisms of action of these compounds were because of the inhibition of tubulin polymerization and induction of a robust G2/M cell-cycle arrest, leading to considerable accumulation of tumor cells in the M-phase. Finally, EAPCs induced tumor cell death by apoptotic pathways. The above-mentioned effects were also observed in most soft tissue tumor cell lines and the gastrointestinal stromal tumor cell line investigated. Taken together, our data identify potent antitumor activity of EAPCs in vitro, thus providing a novel scaffold with which to develop potent chemotherapeutic agents for cancer therapy.

Overweight modifies the longitudinal association between uric acid and some components of the metabolic syndrome: The Tromsø Study.

BACKGROUND: Elevated uric acid (UA) is associated with the presence of the metabolic syndrome (MetS). In a prospective cohort study, we assessed whether baseline and longitudinal change in UA were risk factors for development of MetS and its individual components. METHODS: We included 3087 women and 2996 men who had UA measured in the population based Tromsø Study 1994-95. The participants were stratified according to body mass index (BMI). Endpoints were MetS and each component of the syndrome after 7 years, according to the revised National Cholesterol Education Program's Adult Treatment Panel III (NCEP-ATP III) definition. RESULTS: Multiple logistic regression analyses showed that higher baseline UA was associated with higher odds of developing elevated blood pressure in overweight subjects (BMI ≥ 25 kg/m(2), odds ratio [OR] per 59 µmol/L UA increase 1.44, 95 % confidence interval [CI] = 1.17-1.77, P = 0.001), but not in normal-weight subjects (BMI < 25 kg/m(2), P for interaction = 0.04). Overweight also modified the association between baseline UA and the development of elevated fasting glucose (P for interaction = 0.01). UA was a predictor of MetS in all subjects (OR per 59 µmol/L UA increase 1.29, 95 % CI 1.18-1.41, P < 0.001). Furthermore, longitudinal UA change was independently associated with the development of MetS in all subjects (OR per 59 µmol/L UA increase over 7 years 1.28, 95 % CI 1.16-1.42, P < 0.001). CONCLUSION: Increased levels of baseline UA independently predicted development of elevated blood pressure and higher fasting glycemia in the overweight, but not the normal-weight subjects. Baseline UA and longitudinal increase in UA over 7 years was associated with the development of MetS in all subjects. Whether increased UA should be treated differently in normal-weight and overweight persons needs further study.

The Association Between Adiponectin, Serum Uric Acid and Urinary Markers of Renal Damage in the General Population: Cross-Sectional Data from the Tromsø Study.

BACKGROUND/AIMS: Uric acid may cause renal damage, whereas adiponectin in some studies has been reported to have renoprotective properties. The renoprotective role of adiponectin under the influence of hyperuricemia has not been explored. We assessed the cross-sectional association between adiponectin, serum uric acid (SUA) and urinary biomarkers of glomerular and tubular damage (albumin-creatinine ratio [ACR] and N-acetyl-ß-D-glucosaminidase-creatinine ratio [NAG-CR]) in a large cohort from a general population. METHODS: Three urine specimens from 7062 persons, participating in the Tromsø Study, were collected. The adjusted associations between adiponectin and SUA as independent variables, and ACR ≥1.13 mg/mmol (albuminuria) and the upper gender specific 15 percentile of NAG-CR (high NAG-CR) as dependent variables, were assessed. RESULTS: Mean (standard deviation) age of the participants was 63.5 (9.2) years. Adiponectin was positively associated with albuminuria and high NAG-CR. SUA was associated with albuminuria (odds ratio [OR] 1.13; 95% Confidence Interval [CI] 1.05-1.21 per 59 µmol/L increase), but not with NAG-CR. There were no statistically significant interactions between SUA and adiponectin. CONCLUSIONS: Unexpectedly, adiponectin was positively associated with both urinary markers of renal damage. SUA was positively associated with albuminuria only. SUA and adiponectin added little beyond traditional cardiovascular risk factors to predict renal damage and did not interact in their associations with the urinary biomarkers. Longitudinal studies are needed before firm conclusions can be made.

Cross-sectional analysis of nutrition and serum uric acid in two Caucasian cohorts: the AusDiab Study and the Tromsø study.

BACKGROUND: Hyperuricemia can lead to gout, and may be a risk factor for cardiovascular events, hypertension, diabetes and renal disease. There is well-known link between gout and habitual intake of meat and seafood, however the association between hyperuricemia and micro-and macro-nutrient intake has not been established. METHODS: We studied associations between intakes of food categories, macro-and micronutrients and serum uric acid (SUA) levels in two cross-sectional surveys of Caucasian adults deriving from different food traditions: Australian Diabetes, Obesity and Lifestyle Study 1999/00 (n=9734, age 25-91) and Tromsø Study 4 1994/95 (n = 3031, age 25-69). Dietary intake was calculated from self-administered Food Frequency Questionnaires. In some analyses we stratified according to abdominal obesity status and gender. RESULTS: In both cohorts, lower levels of SUA were found in subjects with higher consumption of carbohydrates, calcium and vitamin B2, while higher fat intake was associated with higher SUA, after adjustment for age, body mass index, estimated glomerular filtration rate, physical activity, total energy intake, use of diuretics, presence of hypertension, diabetes and gout. Among individual food items, high consumption of dairy products, high-fibre bread, cereals and fruits were associated with lower SUA in most subject groups while consumption of meat, eggs, beer and spirits, but not wine, with elevated levels. CONCLUSIONS: Healthy food choices with high intake of carbohydrates, dairy products, fiber and micronutrient-rich foods, and limited intake of fat, beer and spirits, might be recommended to prevent high SUA. Dietary factors seem to have qualitatively similar impact on SUA in obese and non-obese men and women from Australia and Norway.

Uric acid is a risk factor for ischemic stroke and all-cause mortality in the general population: a gender specific analysis from The Tromsø Study.

BACKGROUND: The role of serum uric acid as an independent predictor of cardiovascular disease and death is uncertain in the general population. Adjustments for additional cardiovascular risk factors have not been consistent. We examined the association of serum uric acid with all-cause mortality, ischemic stroke and myocardial infarction in a prospective population based study, with several traditional and non-traditional risk factors for cardiovascular disease included in the model. METHODS: A population-based prospective cohort study was performed among 2696 men and 3004 women. Endpoints were all-cause mortality after 15 years, and fatal or non-fatal myocardial infarction (MI) and ischemic stroke after 12 years. RESULTS: 1433 deaths, 659 MIs and 430 ischemic strokes occurred during follow-up. Fully adjusted Cox regression analyses showed that per 1 SD (87 µmol/L) increase in serum uric acid level, the risk of all-cause mortality increased in both genders (hazard ratios, HR men; 1.11, 95% CI 1.02-1.20, women; 1.16, 1.05-1.29). HRs and 95% CI for stroke were 1.31, 1.14-1.50 in men, 1.13, 0.94-1.36 in women, and 1.22 (1.09, 1.35) in the overall population. No independent associations were observed with MI. CONCLUSION: Serum uric acid was associated with all-cause mortality in men and women, even after adjustment for blood pressure, estimated GFR, urinary albumin/creatinine ratio, drug intake and traditional cardiovascular risk factors. After the same adjustments, serum uric acid was associated with 31% increased risk of stroke in men.

Large method differences for free thyroid hormone assays in the hyperthyroid range can affect assessment of hyperthyroid status: Comparison of Abbott Alinity to Roche Cobas, Siemens Centaur and equilibrium dialysis LC-MS/MS.

BACKGROUND: Free T4 (FT4) determination is one of the most commonly performed biochemical tests in endocrinology. Treatment of thyroid dysfunctions is adjusted based on the severity of symptoms and biochemical test results. For Graves' hyperthyroidism, clinical guidelines recommend using FT4 as a (rough) guide to dose antithyroid drugs, together with other clinical information. It is well known that different platforms and methods give different FT4 results; however, large non-linear method differences at high FT4 concentrations are less well recognized. Current clinical guidelines do not make it clear that method differences in the hyperthyroid range can affect recommendations. METHOD: Serum samples from patients with very low (biochemically hypothyroid) to very high (hyperthyroid) concentrations of FT4 and/or free T3 (FT3) were analyzed using Abbott Alinity and compared to concentrations measured using Roche Cobas, Siemens ADVIA Centaur (FT4 only) and an in-house equilibrium dialysis liquid chromatography tandem mass spectrometry (LC-MS/MS) method. RESULTS: Alinity measured markedly lower FT4 and FT3 concentrations compared to the other methods, particularly at high FT4 concentrations. Regression analysis indicated that Alinity FT4 had a non-linear (curved) relationship to FT4 measured by the other methods. The method differences affected guideline-recommended treatments for hyperthyroidism. CONCLUSION: Measured free thyroid hormone concentrations are highly method-dependent, especially at high FT4 concentrations. Clinicians treating hyperthyroid patients should be aware that patients appear much less hyperthyroid from FT4-measurements performed using Alinity compared to Cobas or Centaur. Guideline-recommended antithyroid drug dosages based on FT4 (including multiples of the upper reference range) have to be adjusted to the FT4 method used. FT4 results from different methods should be clearly distinguished (e.g. separate lines) in medical records.

The association between infant salivary cortisol and parental presence in the neonatal intensive care unit during and after COVID-19 visitation restrictions: A cross-sectional study.

OBJECTIVES: Parent-infant interaction in the neonatal intensive care unit (NICU) promotes health and reduces infant stress. During the COVID-19 pandemic, however, NICUs restricted parent-infant interaction to reduce viral transmission. This study examined the potential relationship between pandemic visitation restrictions, parental presence and infant stress as measured by salivary cortisol. METHODS: A two-NICU cross-sectional study of infants with gestational age (GA) 23-41 weeks, both during (n = 34) and after (n = 38) visitation restrictions. We analysed parental presence with and without visitation restrictions. The relationship between infant salivary cortisol and self-reported parental NICU presence in hours per day was analysed using Pearson's r. A linear regression analysis included potential confounders, including GA and proxies for infant morbidity. The unstandardised B coefficient described the expected change in log-transformed salivary cortisol per unit change in each predictor variable. RESULTS: Included infants had a mean (standard deviation) GA of 31(5) weeks. Both maternal and paternal NICU presence was lower with versus without visitation restrictions (both p ≤0.05). Log-transformed infant salivary cortisol correlated negatively with hours of parental presence (r = -0.40, p = .01). In the linear regression, GA (B = -0.03, p = .02) and central venous lines (B = 0.23, p = .04) contributed to the variance in salivary cortisol in addition to parental presence (B = -0.04 p = .04). CONCLUSION: COVID-19-related visitation restrictions reduced NICU parent-infant interaction and may have increased infant stress. Low GA and central venous lines were associated with higher salivary cortisol. The interaction between immaturity, morbidity and parental presence was not within the scope of this study and merits further investigation.

Metabolomics to Diagnose Oxidative Stress in Perinatal Asphyxia: Towards a Non-Invasive Approach.

There is a need for feasible and non-invasive diagnostics in perinatal asphyxia. Metabolomics is the study of small molecular weight products of cellular metabolism that may, directly and indirectly, reflect the level of oxidative stress. Saliva analysis is a novel approach that has a yet unexplored potential in metabolomics in perinatal asphyxia. The aim of this review was to give an overview of metabolomics studies of oxidative stress in perinatal asphyxia, particularly searching for studies analyzing non-invasively collected biofluids including saliva. We searched the databases PubMed/Medline and included 11 original human and 4 animal studies. In perinatal asphyxia, whole blood, plasma, and urine are the most frequently used biofluids used for metabolomics analyses. Although changes in oxidative stress-related salivary metabolites have been reported in adults, the utility of this approach in perinatal asphyxia has not yet been explored. Human and animal studies indicate that, in addition to antioxidant enzymes, succinate and hypoxanthine, as well acylcarnitines may have discriminatory diagnostic and prognostic properties in perinatal asphyxia. Researchers may utilize the accumulating evidence of discriminatory metabolic patterns in perinatal asphyxia to develop bedside methods to measure oxidative stress metabolites in perinatal asphyxia. Although only supported by indirect evidence, saliva might be a candidate biofluid for such point-of-care diagnostics.

Progression of murine lupus nephritis is linked to acquired renal Dnase1 deficiency and not to up-regulated apoptosis.

The accumulation of apoptotic cells has been suggested as a possible mechanism of nucleosome conversion into self-antigens that may both initiate autoimmune responses and participate in immune complex deposition in lupus nephritis. In this study, we analyzed both the rate of transcription of apoptosis-related genes and the presence of activated apoptotic factors within kidneys of lupus-prone (NZBxNZW) F1 mice during disease progression. The results of this study demonstrated no activation of apoptotic pathways in kidneys of these lupus-prone mice at the time of appearance of anti-double standard DNA antibodies in serum, as well as the formation of mesangial immune deposits in glomeruli. In contrast, the transition of mesangial into membranoproliferative lupus nephritis coincided with an accumulation of activated caspase 3-positive cells in kidneys, in addition to a dramatic decrease in Dnase1 gene transcription. Highly reduced expression levels of the Dnase1 gene may be responsible for the accumulation of large chromatin-containing immune complexes in glomerular capillary membranes. Thus, the initiation of lupus nephritis is not linked to increased apoptotic activity in kidneys. The combined down-regulation of Dnase1 and the increased number of apoptotic cells, which is possibly due to their reduced clearance in affected kidneys, may together be responsible for the transformation of mild mesangial lupus nephritis into severe membranoproliferative, end-stage organ disease.

Crystallized but not soluble uric acid elicits pro-inflammatory response in short-term whole blood cultures from healthy men.

Several epidemiological studies have pointed at serum uric acid (SUA) as an independent risk factor for mortality, diabetes, hypertension, cardiovascular and kidney disease; however, no clear pathogenic pathway is established. Uric acid (UA) crystals show pro-inflammatory properties and can thus create or contribute to the state of chronic low-grade inflammation, a widely accepted pathogenic mechanism in several of the above-mentioned pathologies. On the other hand, soluble uric acid possesses antioxidant properties that might attenuate inflammatory responses. We aimed to explore the net effects of experimentally rising SUA in human whole blood cultures on several mediators of inflammation. Production of TNF-α, IL-1ß, IL-1RA, MCP-1 and IL-8 was assessed upon addition of 200 µM UA, 500 µM UA or monosodium urate (MSU) crystals in the presence or absence of 5 ng/ml lipopolysaccharide (LPS). RT-qPCR and multiplex bead based immunoassay were used to measure mRNA expression and cytokine release at 2 and 4 h of culture, respectively. 14C labeled UA was used to assess intracellular uptake of UA. We show that crystallized, but not soluble, UA induces production of pro-inflammatory mediators in human whole blood. Soluble UA is internalized in blood cells but does not potentiate or reduce LPS-induced release of cytokines.

Increased glomerular matrix metalloproteinase activity in murine lupus nephritis.

Lupus nephritis is associated with thickening of the glomerular basement membrane. Here we measured expression of proteins involved in extracellular matrix turnover in kidneys of lupus-prone mice of the NZBxNZW F1 (B/W) strain before the onset of the disease until the development of proteinuria. Expression of the major isoforms of glomerular basement collagen IV (alpha3/alpha4/alpha5) was unchanged throughout disease progression. Collagen IV alpha1 and alpha2, however, were highly upregulated at the proteinuric stage while collagen IV alpha6 was increased at all time points compared to normal mice. There was increased expression of matrix metalloproteinase-2 and -9, their protein inhibitors TIMP-1 and -2 and the metalloproteinase-9 stabilizing protein lipocalin-2 in kidneys of nephritic lupus-prone mice. When proteinuria appeared we found an increased net glomerular gelatinolytic activity. These studies suggest that matrix metalloproteinases contribute to extracellular matrix expansion and proteinuria by altering matrix composition.

Effects of CpG-oligonucleotides, poly I:C and LPS on Atlantic cod scavenger endothelial cells (SEC).

Carrying out a remarkably efficient clearance of colloidal and soluble macromolecular waste substances from the circulation, the scavenger endothelial cells (SECs) represent an important part of the reticuloendothelial system of vertebrates. It has been previously shown that these cells play an important role in the innate immune system by eliminating from the blood a number of molecules known to elicit inflammatory reactions. In the present study we have investigated the uptake of LPS and oligonucleotides in cultured Atlantic cod SECs, and determined if interaction with these pathogen associated molecules affect the scavenger activity and/or production of immune modulating molecules of SECs. Preincubation of cultured SECs with CpG (5 and 20 microg/ml) or Poly I:C (10 or 40 microg/ml) gave selective down-regulation of scavenger receptor-mediated endocytosis, but only marginal effects were noted on endocytosis via the mannose- and collagen alpha chain receptors. Preincubation with LPS or a non-inflammatory ligand for the scavenger receptor did not result in altered endocytosis via any of the receptors tested. Only Poly I:C (40 microg/ml) was observed to increase the production of NO. RT-PCR analyses showed IL-1 production which was not increased above control after pre-treatment with two different CpG-oligonucleotides or Poly I:C. In fact, preincubation with Poly I:C, but not CpGs, resulted in degradation of total intracellular RNA. In conclusion, our study shows that SECs respond differently to the different immunomodulators used and that their important clearance activity as scavenger cells can be regulated by the use of oligonucleotides.

Glomerular targets for autoantibodies in lupus nephritis--an apoptotic origin.

Systemic lupus erythematosus (SLE) is an autoimmune syndrome where different organs may individually or simultaneously be affected. Whether SLE is one disease entity or represents a variety of intrinsically unrelated organ manifestations is unknown. Variability of clinical presentations of SLE argues against the former. This does not, however, exclude that certain organ manifestations may be pathogenetically linked. It is believed that in situ binding of anti-dsDNA antibodies by nucleosomes is involved in organ manifestations in SLE. This review will focus on nature and origin of target structures for anti-dsDNA and antinucleosome antibodies in glomerular capillary and mesangial matrix membranes. We will particularly discuss the potential role of apoptosis and release of apoptotic chromatin in terms of their putative impact in SLE.

Macrophage stimulating agent soluble yeast ß-1,3/1,6-glucan as a topical treatment of diabetic foot and leg ulcers: A randomized, double blind, placebo-controlled phase II study.

AIMS/INTRODUCTION: Dysregulated inflammatory response is believed to be an important factor in the pathogenesis of several late complications of diabetes mellitus. ß-Glucans are potent inducers of immune function. The present randomized, double blind, two-center, placebo-controlled study was undertaken to explore safety, tolerability and efficacy of soluble ß-1,3/1,6-glucan (SBG) as a local treatment of diabetic foot ulcers. MATERIALS AND METHODS: A total of 60 patients with type 1 or 2 diabetes and lower extremity ulcers (Wagner grade 1-2, Ankle/Brachial Index ≥0.7) received SBG or a comparator product (methylcellulose) locally three times weekly up to 12 weeks in addition to conventional management scheme. A total of 54 patients completed the study. RESULTS: A tendency for shorter median time to complete healing in the SBG group was observed (36 vs 63 days, P = 0.130). Weekly percentage reduction in ulcer size was significantly higher in the SBG group than in the methylcellulose group between weeks 1-2, 3-4 and 5-6 (P < 0.05). The proportion of ulcers healed by week 12 was also in favor of SBG (59% vs 37%, P = 0.09), with a significantly higher healing incidence in the SBG group at week 8 (44% vs 17%, P = 0.03). SBG was safe and well tolerated. There was a clinically significant difference regarding the incidence of serious adverse events in favor of the SBG treatment. CONCLUSIONS: Local treatment of diabetic lower extremity ulcers with ß-1,3/1,6-polyglucose shows good safety results. This ß-glucan preparation shows promising potential as a treatment accelerating cutaneous healing. Further studies are required to confirm this effect. This trial was registered with ClinicalTrials.gov (no. NCT00288392).