Topical non-steroidal anti-inflammatory drugs for central serous chorioretinopathy: A systematic review and meta-analysis.

Central serous chorioretinopathy (CSC) is a prevalent exudative maculopathy and the ongoing verteporfin shortage restricts current treatment possibilities. Topical non-steroidal anti-inflammatory drugs (NSAID) have previously been proposed as a treatment for CSC, although its exact efficacy remains unclear. In this systematic review and meta-analysis, we outlined the efficacy of topical NSAIDs for the treatment of CSC. We searched 11 literature databases on 13 December 2022, for any study describing topical NSAID treatment for CSC. Thirteen eligible studies were included with a total of 1001 eyes of 994 patients with CSC. Six studies were case reports, two were cohort studies and five were non-randomized comparative studies. Where specified, topical NSAIDs used were bromfenac 0.09%, diclofenac 0.1%, ketorolac 0.4% and 0.5%, pranoprofen 0.1%, and nepafenac 0.1% and 0.3%. Studies were predominantly of cases with acute CSC and several case studies reported treatment outcomes simultaneously with discontinuation of corticosteroid use, which complicated treatment evaluation. Meta-analyses of comparative studies revealed a statistically significant but clinically irrelevant best-corrected visual acuity improvement of -0.04 logMAR (95% CI: -0.07 to -0.01 logMAR; p = 0.01) at 1-month follow-up, which became statistically insignificant at 3-month follow-up (-0.03 logMAR; 95% CI: -0.06 to 0.003 logMAR; p = 0.08). Further, we found no benefit in complete subretinal fluid resolution at 1-month follow-up (OR: 1.20; 95% CI: 0.81-1.76; p = 0.37) or 3-month follow-up (OR: 1.17; 95% CI: 0.86 to 1.59; p = 0.33). Taken together, available evidence does not support the use of topical NSAIDs for the treatment of CSC.

European Prevalence of Polypoidal Choroidal Vasculopathy: A Systematic Review, Meta-Analysis, and Forecasting Study.

The purpose of this study was to provide an estimate of the number of current and future patients with polypoidal choroidal vasculopathy (PCV) in Europe. We systematically searched 11 literature databases on 18 May 2022 for studies on the prevalence of PCV among a consecutive and representative group of patients with suspected neovascular age-related macular degeneration (AMD). Prevalence of PCV in patients with suspected neovascular AMD was summarized and included in a prevalence meta-analysis. We then used current population data and population forecasts by Eurostat and the Office for National Statistics to determine current and future number of patients with neovascular AMD in Europe. Then, we calculated the number of patients with PCV with our calculated estimate of the prevalence of PCV among Europeans suspected with neovascular AMD. A total of five eligible studies were identified which included a total of 1359 patients. All these studies used the gold standard of indocyanine green angiography as a routine part of their diagnostic approach. Among patients undergoing detailed retinal examination for suspected neovascular AMD, our meta-analysis calculated the prevalence of PCV to be 8.3% (95% confidence interval: 6.8-9.8%). Our population estimates find that a total of 217,404 patients with PCV exist in Europe in the year 2022, which constitutes 0.04% of the entire population of Europe. This number is estimated to increase to 287,517 patients in the year 2040. Our estimates are important for different healthcare stakeholders, especially when planning and allocating expensive resources.

Pachychoroid Spectrum Diseases in Patients with Cushing's Syndrome: A Systematic Review with Meta-Analyses.

Cushing's syndrome is a rare disease with an endogenous cause of excess cortisol secretion. More evidence substantially links cortisol levels to the pachychoroid spectrum diseases. In this systematic review and meta-analysis, we summarize available evidence on pachychoroid spectrum diseases in patients with Cushing's syndrome. We performed a systematic literature search in 11 databases on 21 May 2022. Studies were considered eligible if they performed retinal examination of a consecutive group of patients with Cushing's syndrome using optical coherence tomography (OCT) scans. We extracted data on subfoveal choroidal thickness in patients with Cushing's syndrome compared to matched controls. We also extracted data on the prevalence of pachychoroid pigment epitheliopathy (PPE), central serous chorioretinopathy (CSC), and polypoidal choroidal vasculopathy (PCV). We identified six eligible studies with a total of 159 patients with Cushing's syndrome. On average, patients with Cushing's syndrome have 49.5 µm thicker subfoveal choroidal thickness compared to matched healthy individuals. Pachychoroid spectrum diseases were relatively common in these patients: PPE in 20.8%, CSC in 7.7%, and PCV in 2.8%. We conclude that there should be low threshold to recommend ophthalmic examination to patients with Cushing's syndrome, and that a macular OCT is recommended during this examination.

Familial colorectal cancer and tooth agenesis caused by an AXIN2 variant: how do we detect families with rare cancer predisposition syndromes?

We present a three-generation family with an AXIN2 variant and a family history of colorectal cancer (CRC), colon polyps and tooth agenesis. A likely pathogenic variant was detected in the AXIN2 gene (c.1994dup; p.(Asn666Glnfs*41)). This variant has previously been associated with tooth agenesis and polyposis, only. In this case report we describe eight carriers with tooth agenesis and variable clinical findings, including polyps and CRC. Our case provides additional knowledge to the sparse data on genotype-phenotype association related to AXIN2 associated cancer syndrome. Further, our case highlights the importance of analysing an extended CRC and oligodontia/ectodermal dysplasia gene panel including AXIN2 but also raises awareness and discussion about appropriate surveillance program.

Gene-ecology of durum wheat HMW glutenin reflects their diffusion from the center of origin.

The production of many food items processed from wheat grain relies on the use of high gluten strength flours. As a result, about 80% of the allelic variability in the genes encoding the glutenin proteins has been lost in the shift from landraces to modern cultivars. Here, the allelic variability in the genes encoding the high molecular weight glutenin subunits (HMW-GSs) has been characterized in 152 durum wheat lines developed from a set of landraces. The allelic composition at the two Glu-1 loci (Glu-A1 and -B1) was obtained at both the protein and the DNA level. The former locus was represented by three alleles, of which the null allele Glu-A1c was the most common. The Glu-B1 locus was more variable, with fifteen alleles represented, of which Glu-B1b (HMW-GSs 7 + 8), -B1d (6 + 8) and -B1e (20 + 20) were the most frequently occurring. The composition of HMW-GSs has been used to make inferences regarding the diffusion and diversification of durum wheat. The relationships of these allelic frequencies with their geographical distribution within the Mediterranean basin is discussed in terms of gene-ecology.

Changes in urinary excretion of water and sodium transporters during amiloride and bendroflumethiazide treatment.

AIM: To quantify changes in urinary excretion of aquaporin2 water channels (u-AQP2), the sodium-potassium-chloride co-transporter (u-NKCC2) and the epithelial sodium channels (u-ENaC) during treatment with bendroflumethiazide (BFTZ), amiloride and placebo. METHODS: In a randomized, double-blinded, placebo-controlled, 3-way crossover study we examined 23 healthy subjects on a standardized diet and fluid intake. The subjects were treated with amiloride 5 mg, BFTZ 1.25 mg or placebo twice a day for 4.5 d before each examination day. On the examination day, glomerular filtration rate was measured by the constant infusion clearance technique with (51)Cr-EDTA as reference substance. To estimate the changes in water transport via AQP2 and sodium transport via NKCC2 and ENaC, u-NKCC2, the gamma fraction of ENaC (u-ENaCγ), and u-AQP2 were measured at baseline and after infusion with 3% hypertonic saline. U-NKCC2, u-ENaCγ, u-AQP2 and plasma concentrations of vasopressin (p-AVP), renin (PRC), angiotensin II (p-ANG II) and aldosterone (p-Aldo) were measured, by radioimmunoassay. Central blood pressure was estimated by applanation tonometry and body fluid volumes were estimated by bio-impedance spectroscopy. General linear model with repeated measures or related samples Friedman's two-way analysis was used to compare differences. Post hoc Bonferroni correction was used for multiple comparisons of post infusion periods to baseline within each treatment group. RESULTS: At baseline there were no differences in u-NKCC2, u-ENaCγ and u-AQP2. PRC, p-Ang II and p-Aldo were increased during active treatments (P < 0.001). After hypertonic saline, u-NKCC2 increased during amiloride (6% ± 34%; P = 0.081) and increased significantly during placebo (17% ± 24%; P = 0.010). U-AQP2 increased significantly during amiloride (31% ± 22%; P < 0.001) and placebo (34% ± 27%; P < 0.001), while u-NKCC2 and u-AQP2 did not change significantly during BFTZ (-7% ± 28%; P = 0.257 and 5% ± 16%; P = 0.261). U- ENaCγ increased in all three groups (P < 0.050). PRC, AngII and p-Aldo decreased to the same extent, while AVP increased, but to a smaller degree during BFTZ (P = 0.048). cDBP decreased significantly during BFTZ (P < 0.001), but not during amiloride or placebo. There were no significant differences in body fluid volumes. CONCLUSION: After hypertonic saline, u-NKCC2 and u-AQP2 increased during amiloride, but not during BFTZ. Lower p-AVP during BFTZ potentially caused less stimulation of NKCC2 and AQP2 and subsequent lower reabsorption of water and sodium.

Effect of nebivolol on renal nitric oxide availability and tubular function in patients with essential hypertension.

AIMS: Nebivolol is a selective ß1 -receptor antagonist with vasodilating properties. In patients with essential hypertension, we tested the hypothesis that nebivolol increases systemic and renal nitric oxide (NO) availability using L-N(G) -monomethyl arginine (L-NMMA) as an inhibitor of NO production. METHODS: In a randomized, placebo-controlled, crossover study, patients with essential hypertension were treated with nebivolol for five days, along with a standardized diet and fluid intake. We examined the acute effects of systemic NO synthase inhibition with L-NMMA on brachial blood pressure (bBP), pulse wave velocity (PWV) and central blood pressure (cBP) estimated by applanation tonometry, glomerular filtration rate (GFR), fractional excretion of sodium (FENa ), urinary excretion of both aquaporin-2 (u-AQP2) and epithelial sodium channels (u-ENaCγ ), and plasma concentrations of nitrate/nitrite (p-NOx ) and vasoactive hormones after five days' treatment with placebo and nebivolol. RESULTS: Nebivolol significantly reduced PWV, bBP, cBP and plasma renin, angiotensin II and aldosterone concentrations. The renal parameters, p-NOx and plasma arginine vasopressin concentration were not changed by nebivolol. There was no difference between nebivolol and placebo in the response to L-NMMA, with LMMA inducing a similar increase in PWV, bBP and cBP and a similar decrease in GFR, uAQP2 and u-ENaCγ and FENa [mean change -0.62% (95% confidence interval {CI} -0.40 to -0.84) during placebo vs. -0.57% (95% CI -0.46 to -0.68; P = 0.564) during nebivolol treatment]. Vasoactive hormones were changed to a similar extend by L-NMMA during administration of nebivolol and placebo. CONCLUSIONS: Nebivolol did not change p-NOx , and inhibition of NO synthesis induced the same response in blood pressure, GFR, renal tubular function and vasoactive hormones during nebivolol and placebo. Thus, the data did not support the hypothesis that nebivolol changes vascular and renal NO availability in patients with essential hypertension.

Abnormal urinary excretion of NKCC2 and AQP2 in response to hypertonic saline in chronic kidney disease: an intervention study in patients with chronic kidney disease and healthy controls.

BACKGROUND: Renal handling of sodium and water is abnormal in chronic kidney disease (CKD). The aim of this study was to test the hypothesis that abnormal activity of the aquaporin-2 water channels (AQP2), the sodium-potassium-2chloride transporter (NKCC2) and/or the epithelial sodium channels (ENaC) contribute to this phenomenon. METHODS: 23 patients with CKD and 24 healthy controls at baseline and after 3% saline infusion were compared. The following measurements were performed: urinary concentrations of AQP2 (u-AQP2), NKCC2 (u-NKCC2), ENaC (u-ENaCγ), glomerular filtration rate (GFR) estimated by 51Cr-EDTA clearance, free water clearance (CH2O), urinary output (UO), fractional excretion of sodium (FENa), plasma concentrations of AVP, renin (PRC), Angiotensin II (ANG II), Aldosterone (Aldo) and body fluid volumes. RESULTS: At baseline, GFR was 34 ml/min in CKD patients and 89 ml/ml in controls. There were no significant differences in u-AQP2, u-NKCC2 or u-ENaCγ, but FENa, p-Aldo and p-AVP were higher in CKD patients than controls. In response to hypertonic saline, patients with CKD had an attenuated decrease in CH2O and UO. A greater increase in U-AQP2 was observed in CKD patients compared to controls. Furthermore, u-NKCC2 increased in CKD patients, whereas u-NKCC2 decreased in controls. Body fluid volumes did not significantly differ. CONCLUSIONS: In response to hypertonic saline, u-NKCC2 increased, suggesting an increased sodium reabsorption via NKCC2 in patients with CKD. U-AQP2 increased more in CKD patients, despite an attenuated decrease in CH2O. Thus, though high levels of p-AVP and p-Aldo, the kidneys can only partly compensate and counteract acute volume expansion due to a defective tubular response. TRIAL REGISTRATION: Clinical trial no: NCT01623661. Date of trial registration: 18.06.2012.

Effect of atorvastatin on renal NO availability and tubular function in patients with stage II-III chronic kidney disease and type 2 diabetes.

BACKGROUND: Statins have beneficial effects on cardiovascular morbidity and mortality independently of reduction of plasma cholesterol. PURPOSE AND METHODS: In patients with type 2 diabetes and nephropathy, chronic kidney disease stage II-III, we tested the hypothesis that atorvastatin increased systemic and renal nitric oxide (NO) availability using L-NMMA as an inhibitor of NO production. We performed a randomized, placebo-controlled, crossover study, using atorvastatin/placebo treatment for five days with a standardized diet and fluid intake. We measured brachial BP (bBP), central BP (cBP), GFR, urinary output (OU), free water clearance (CH2O), fractional excretion of sodium (FENa), urinary excretion of albumin (UAER and UACR), AQP2 (u-AQP2) and ENaC (u-ENaCγ) and plasma concentrations of vasoactive hormones: renin, angiotensin II, aldosterone, arginine vasopressin, endothelin-1 and brain natriuretic peptide. RESULTS: During atorvastatin and placebo treatment, L-NMMA infusion, changed the effect variables significantly, but to the same extent, i.e. an increase in bBP and cBP, and a decrease in GFR, OU, CH2O, FENa, u-AQP2 and u-ENaCγ. In addition, renin and angiotensin II was reduced, aldosterone increased, and vasopressin, endothelin-1 and brain natriuretic hormone unchanged. CONCLUSION: During, atorvastatin and placebo treatment, inhibition of nitric oxide synthesis induced the same response in brachial and central blood pressure, GFR, renal tubular function and vasoactive hormones. Thus, atorvastatin did not change nitric oxide availability in type 2 diabetics with nephropathy.

Effect of volume expansion with hypertonic- and isotonic saline and isotonic glucose on sodium and water transport in the principal cells in the kidney.

BACKGROUND: The renal distal nephron plays an important role in the maintenance of sodium balance, extra cellular volume and blood pressure. The degree of water transport, via aquaporin2 water channels (AQP2), and sodium transport, via epithelial sodium channels (ENaC) in renal collecting duct principal cells are reflected by the level of urinary excretion of AQP2 (u-AQP2) and the γ-fraction of ENaC (u-ENaCγ). The effects of an acute intravenous volume load with isotonic saline, hypertonic saline and glucose on u-AQP2, u-ENaCγ and underlying mechanisms have never been studied in a randomized, placebo-controlled trial in healthy humans. METHODS: We studied the effects of 0.9% saline (23 ml/kg), 3% saline (7 ml/kg) and 5% glucose (23 ml/kg) on u-AQP2 and u-ENaCγ, fractional sodium excretion (FENa), free water clearance (CH2O), and plasma concentrations of vasopressin (AVP), renin (PRC), angiotensin II (ANG II) and aldosterone (Aldo) in a randomized, crossover study of 23 healthy subjects, who consumed a standardized diet, regarding calories, sodium and fluid for 4 days before each examination day. RESULTS: After isotonic saline infusion, u-AQP2 increased (27%). CH2O and u-ENaCγ were unchanged, whereas FENa increased (123%). After hypertonic saline infusion, there was an increase in u-AQP2 (25%), u-ENaCγ (19%) and FENa (96%), whereas CH2O decreased (-153%). After isotonic glucose infusion, there was a decrease in u-AQP2 (-16%), ENaCγ (-10%) and FENa (-44%) whereas CH2O increased (164%). AVP remained unchanged after isotonic saline and glucose, but increased after hypertonic saline (139%). PRC, AngII and p-Aldo decreased after isotonic and hypertonic saline infusion, but not after glucose infusion. CONCLUSIONS: Volume expansion with 3% and 0.9% saline increased u-AQP2, while isotonic glucose decreased u-AQP2. Infusion of hypertonic saline increased u-ENaCγ, whereas u-ENaCγ was not significantly changed after isotonic saline and tended to decrease after glucose. Thus, the transport of water and sodium is changed both via the aquaporin 2 water channels and the epithelial sodium channels during all three types of volume expansion to regulate and maintain water- and sodium homeostasis in the body. TRIAL REGISTRATION: Clinical Trial no: NCT01414088.

Inhibition of p53-dependent, but not p53-independent, cell death by U19 protein from human herpesvirus 6B.

Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after γ irradiation in both permissive and non-permissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following γ irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein.

Functional characterisation of wheat Pgip genes reveals their involvement in the local response to wounding.

Polygalacturonase-inhibiting proteins (PGIPs) are cell wall leucine-rich repeat (LRR) proteins involved in plant defence. The hexaploid wheat (Triticum aestivum, genome AABBDD) genome contains one Pgip gene per genome. Tapgip1 (B genome) and Tapgip2 (D genome) are expressed in all tissues, whereas Tapgip3 (A genome) is inactive because of a long terminal repeat, Copia retrotransposon insertion within the coding region. To verify whether Tapgip1 and Tapgip2 encode active PGIPs and are involved in the wheat defence response, we expressed them transiently and analysed their expression under stress conditions. Neither TaPGIP1 nor TaPGIP2 showed inhibition activity in vitro against fungal polygalacturonases. Moreover, a wheat genotype (T. turgidum ssp. dicoccoides) lacking active homologues of Tapgip1 or Tapgip2 possesses PGIP activity. At transcript level, Tapgip1 and Tapgip2 were both up-regulated after fungal infection and strongly induced following wounding. This latter result has been confirmed in transgenic wheat plants expressing the ß-glucuronidase (GUS) gene under control of the 5'-flanking region of Tdpgip1, a homologue of Tapgip1 with an identical sequence. Strong and transient GUS staining was mainly restricted to the damaged tissues and was not observed in adjacent tissues. Taken together, these results suggest that Tapgips and their homologues are involved in the wheat defence response by acting at the site of the lesion caused by pathogen infection.

Effect of amiloride and spironolactone on renal tubular function and central blood pressure in patients with arterial hypertension during baseline conditions and after furosemide: a double-blinded, randomized, placebo-controlled crossover trial.

This study demonstrates that the increased potassium content in the body seems to change both the blood pressure and renal tubular function. We wanted to test the hypotheses that amiloride and spironolactone induced potassium retention reduces ambulatory blood pressure (ABP) and central blood pressure (CBP) during baseline conditions and after furosemide and that the tubular transport via the epithelial sodium channels (ENaCs) and aquaporin-2 (AQP2) water channels was increased by furosemide in arterial hypertension. Each of three 28-day treatment periods (placebo, amiloride, and spironolactone) was completed by a 4-day period with standardized diet regarding calories and sodium and water intake. At the end of each period, we measured pulse wave velocity (PWV), central systolic blood pressure (CSBP), central diastolic blood pressure (CDBP), glomerular filtration rate (GFR), free water clearance (CH2O), fractional excretion of sodium (FENa) and potassium (FEK), urinary excretion of AQP2 (u-AQP2), urinary excretion of γ-fraction of the ENaC (u-ENaCγ), and plasma concentrations of renin (PRC), angiotensin II (p-Ang II), and aldosterone (p-Aldo) at baseline conditions and after furosemide bolus. Ambulatory blood pressure and CBP were significantly lowered by amiloride and spironolactone. During 24-hour urine collection and at baseline, GFR, CH2O, FENa, FEK, u-AQP2 and u-ENaCγ were the same. After furosemide, CH2O, FENa, FEK, u-AQP2, u-ENaCγ, PRC, p-Ang II, p-Aldo, PWV and CDBP increased after all treatments. However, during amiloride treatment, FEK increased to a larger extent than after spironolactone and during placebo after furosemide, and CSBP was not significantly reduced. The increases in water and sodium absorption via AQP2 and ENaC after furosemide most likely are compensatory phenomena to antagonize water and sodium depletion. Amiloride is less effective than spironolactone to reduce renal potassium excretion.

Intron retention regulates the expression of pectin methyl esterase inhibitor (Pmei) genes during wheat growth and development.

Pectin is an important component of the plant cell wall and its remodelling occurs during normal plant growth or following stress responses. Pectin is secreted into the cell wall in a highly methyl-esterified form and subsequently de-methyl-esterified by pectin methyl esterase (PME), whose activity is controlled by the pectin methyl esterase inhibitor protein (PMEI). Cereal cell wall contains a low amount of pectin; nonetheless the level and pattern of pectin methyl esterification play a primary role during development or pathogen infection. Since few data are available on the role of PMEI in plant development and defence of cereal species, we isolated and characterised three Pmei genes (Tdpmei2.1, Tdpmei2.2 and Tdpmei3) and their encoded products in wheat. Sequence comparisons showed a low level of intra- and inter-specific sequence conservation of PMEIs. Tdpmei2.1 and Tdpmei2.2 share 94% identity at protein level, but only 20% identity with the product of Tdpmei3. All three Tdpmei genes code for functional inhibitors of plant PMEs and do not inhibit microbial PMEs or a plant invertase. RT-PCR analyses demonstrated, for the first time to our knowledge, that Pmei genes are regulated by intron retention. Processed and unprocessed transcripts of Tdpmei2.1 and Tdpmei2.2 accumulated in several organs, but anthers contained only mature transcripts. Tdpmei3 lacks introns and its transcript accumulated mainly in stem internodes. These findings suggest that products encoded by these Tdpmei genes control organ- or tissue-specific activity of specific PME isoforms in wheat.

Transgenic expression of polygalacturonase-inhibiting proteins in Arabidopsis and wheat increases resistance to the flower pathogen Fusarium graminearum.

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most important diseases of wheat worldwide, resulting in yield losses and mycotoxin contamination. The molecular mechanisms regulating Fusarium penetration and infection are poorly understood. Beside mycotoxin production, cell wall degradation may play a role in the development of FHB. Many fungal pathogens secrete polygalacturonases (PGs) during the early stages of infection, and plants have evolved polygalacturonase-inhibiting proteins (PGIPs) to restrict pectin degradation during fungal infection. To investigate the role of plant PGIPs in restricting the development of FHB symptoms, we first used Arabidopsis thaliana, whose genome encodes two PGIPs (AtPGIP1 and AtPGIP2). Arabidopsis transgenic plants expressing either of these PGIPs under control of the CaMV 35S promoter accumulate inhibitory activity against F. graminearum PG in their inflorescences, and show increased resistance to FHB. Second, transgenic wheat plants expressing the bean PvPGIP2 in their flowers also had a significant reduction of symptoms when infected with F. graminearum. Our data suggest that PGs likely play a role in F. graminearum infection of floral tissues, and that PGIPs incorporated into wheat may be important for increased resistance to FHB.

Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies.

Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface loops that undergo conformational changes during zymogen activation.

Direct Repeat 6 from human herpesvirus-6B encodes a nuclear protein that forms a complex with the viral DNA processivity factor p41.

The SalI-L fragment from human herpesvirus 6A (HHV-6A) encodes a protein DR7 that has been reported to produce fibrosarcomas when injected into nude mice, to transform NIH3T3 cells, and to interact with and inhibit the function of p53. The homologous gene in HHV-6B is dr6. Since p53 is deregulated in both HHV-6A and -6B, we characterized the expression of dr6 mRNA and the localization of the translated protein during HHV-6B infection of HCT116 cells. Expression of mRNA from dr6 was inhibited by cycloheximide and partly by phosphonoacetic acid, a known characteristic of herpesvirus early/late genes. DR6 could be detected as a nuclear protein at 24 hpi and accumulated to high levels at 48 and 72 hpi. DR6 located in dots resembling viral replication compartments. Furthermore, a novel interaction between DR6 and the viral DNA processivity factor, p41, could be detected by confocal microscopy and by co-immunoprecipitation analysis. In contrast, DR6 and p53 were found at distinct subcellular locations. Together, our data imply a novel function of DR6 during HHV-6B replication.

A novel mode of intervention with serine protease activity: targeting zymogen activation.

Serine proteases are secreted from cells as single-chain zymogens, typically having activities orders of magnitude lower than those of the mature two-chain enzymes. Activation occurs by a conformational change initiated by cleavage of a specific peptide bond. We have derived a monoclonal antibody (mAb-112) which binds with subnanomolar affinity to pro-uPA, the zymogen form of urokinase-type plasminogen activator (uPA). We mapped the epitope of the antibody to the autolysis loop, one of the structural elements known to change conformation during zymogen activation. A mechanistic evaluation with biophysical methods elucidated a novel bifunctional inhibitory mechanism whereby mAb-112 not only delays the proteolytic conversion of single-chain pro-uPA into the two-chain form but also subsequently averts the conformational transition to a mature protease by sequestering the two-chain form in a zymogen-like, noncatalytic state. Functional studies employing two variants of human HT-1080 cells, exhibiting high and low levels of dissemination in a chorioallantoic membrane assay, demonstrate that mAb-112 is an effective inhibitor of tumor cell intravasation. These findings show that pharmacological interference with zymogen activation is a plausible and robust means to regulate uPA activity and the downstream effects of plasminogen activation in the spread of cancer and other processes of pathological tissue remodeling. A strategy that targets regions related to pro-enzyme activation likely provide a unique inhibitor-protease interaction surface and is, thus, expected to enhance the chances of engineering high inhibitor specificity. Our results provide new information about the structural flexibility underlying the equilibrium between active and inactive forms of serine proteases.