Influence of spontaneous platelet aggregation on progression of glomerular disease

Platelet secretion products may play an important role in the pathogenesis and progression of the kidney disease. Amongst the parameters describing platelet hyperactivity the measurement of spontaneous platelet aggregation (SPA) seems particularly useful. In this study SPA as well as mean platelet volume (MPV), modal platelet volume (PLT Mode) and platelet count (PLT) were investigated in 60 patients with biopsy proven primary glomerulonephritis. SPA was measured using the turbidimetric method according to Born with no enhancers added. Serum creatinine concentration (Cr), reciprocal serum creatinine concentration (1/Cr) and endogenous creatinine clearance (Cl(Cr)) were used for the renal function estimation. Protein and lipid profiles as well as coagulo-fibrinolytic balance were measured in parallel. The investigated group consisted of 30 non-nephrotic patients (CGN) - in 9, SPA was found (CGN-B) while 21 had SPA <10% (CGN-A), and 30 nephrotic patients (CGN+NS) - 19 with SPA (CGN+NS-B) and 11 without (CGN+NS-A). SPA was found to be a constant platelet feature in patients with chronic glomerulopathy. The group remained under observation for 36 months. 41 patients were included in the 3-year prospective study which revealed the significant influence of the blood platelet hyperaggregability on the renal disease progression. A significantly increased serum creatinine concentration, decreased 1/Cr parameter and decreased glomerular filtration rate (Cl(Cr)) were noted in subgroups showing SPA. A significant correlation between SPA and (Delta)Cr/month (r = 0.41), (Delta)1/Cr/month (r = 0.38) as well as (Delta)Cl(Cr)/month (r = 0.52) was found. The platelet activity and thus SPA can be altered by various factors: albumin and fibrinogen plasma concentrations, thrombosis activation and possibly lipoprotein metabolism disturbances. A characteristic feature of spontaneously aggregating platelet is their increased volume (MPV). CONCLUSION: Platelet hyperaggregation in one of nonimmunological factors stimulates the progression of glomerulonephritis.     

Phase I trial of a novel matrix metalloproteinase inhibitor batimastat (BB-94) in patients with advanced cancer

Summary Degradation of basement membrane and extracellular matrix by matrix metalloproteinases (MMPs) is believed to be required for tumor invasion, tumor-induced angiogenesis and vascular invasion. A synthetic hydroxamate, batimastat (also known as BB-94), inhibits MMPs by binding the zinc ion in the active site of the MMP. Batimastat inhibits at least 50% of MMP activity at concentrations less than or equal to 10 ng/ml in vitro. Batimastat retarded ascites accumulation and increased survival in mice with human ovarian tumor xenografts. Acute and long-term toxicological studies revealed no major toxicity in animals. Batimastat is poorly soluble and was administered intraperitoneally (i.p.) as a suspension. Previous studies in patients with malignant ascites have shown no major toxicities at doses as high as 1350 mg/m².     

Distinct molecular phenotype of malignant CD34(+) hematopoietic stem and progenitor cells in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a malignant disorder of the hematopoietic stem cell characterized by the BCR-ABL oncogene. We examined gene expression profiles of highly enriched CD34(+) hematopoietic stem and progenitor cells from patients with CML in chronic phase using cDNA arrays covering 1.185 genes. Comparing CML CD34(+) cells with normal CD34(+) cells, we found 158 genes which were significantly differentially expressed. Gene expression patterns reflected BCR-ABL-induced functional alterations such as increased cell-cycle and proteasome activity. Detoxification enzymes and DNA repair proteins were downregulated in CML CD34(+) cells, which might contribute to genetic instability. Decreased expression of junction plakoglobulin and CXC chemokine receptor 4 (CXCR-4) might facilitate the release of immature precursors from bone marrow in CML. GATA-2 was upregulated in CML CD34(+) cells, suggesting an increased self-renewal in comparison with normal CD34(+) cells. Moreover, we found upregulation of the proto-oncogene SKI and of receptors for neuromediators such as opioid mu1 receptor, GABA B receptor, adenosine A1 receptor, orexin 1 and 2 receptors and corticotropine-releasing hormone receptor. Treatment of CML progenitor cells with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) resulted in a dose-dependent significant inhibition of clonogenic growth by 40% at a concentration of 10(-5) M, which could be reversed by the equimolar addition of the receptor agonist 2-chloro-N6-cyclopentyladenosine (P<0.05). The incubation of normal progenitor cells with DPCPX resulted in an inhibition of clonogenic growth to a significantly lesser extent in comparison with CML cells (P<0.05), suggesting that the adenosine A1 receptor is of functional relevance in CML hematopoietic progenitor cells.     

Circulating soluble tumour necrosis factor receptors in patients with epidermodysplasia verruciformis as compared to patients with cutaneous tumours in the general population

Soluble tumour necrosis factor receptors type I and II (sTNF-RI and II) were evaluated in sera from patients with epidermodysplasia verruciformis and patients with cutaneous warts, actinic keratoses, squamous cell carcinomas or basal cell carcinomas by specific enzyme-linked immunobiological assays. In patients with widespread epidermodysplasia verruciformis lesions, the levels of both sTNF-Rs were in normal range. Both types of sTNF-Rs were significantly increased in patients with warts. The levels of sTNF-RI were significantly increased in patients with multiple actinic keratoses, squamous cell carcinoma and basal cell carcinoma. Increased levels of circulating sTNF-Rs may facilitate development of cutaneous tumours. Normal levels of sTNF-Rs in patients with epidermodysplasia verruciformis might, at least partially, contribute to a slow growth and low metastatic potential of cancers in these patients.     

Expression of insulin-like growth factor-I receptor, estrogen receptor alpha, Bcl-2 and Bax proteins in human breast cancer

Disturbance in expression of estrogen receptors together with changing influence of growth factor receptors and apoptosis associated proteins plays a role in breast cancer development and progression. However, immunohistochemical detection and relationships among these proteins were not often considered in relation to breast cancer and a few evaluations of expression provided mismatching results and conclusions. Consequently, we examined by immunohistochemistry the expression of the insulin-like growth factor-I receptor (IGF-IR), estrogen receptor alpha (ERalpha) and apoptosis-associated proteins, Bcl-2 and Bax, in human primary breast cancer, as well as analyzing the relationships among these proteins. The positive immunostaining for IGF-IR, ERalpha, Bcl-2 and Bax was noted in 56, 63.8, 82.8 and 50% of tumors, respectively. We observed that IGF-IR negatively correlated with ERalpha in the group of all tumors and in axillary node negative cancer (p<0.03, p<0.05, respectively), but not in the subgroup of node positive cancer. Expression of ERalpha correlated positively with Bcl-2 and negatively with Bax proteins (p<0.0001, p<0.05, respectively). We did not note significant relationships between IGF-IR and Bcl-2, or IGF-IR and Bax proteins. We found that increased Bax expression was associated with positive lymph node status, pT2 stage and G3 grade of tumors. Knowledge about alterations in the IGF-IR expression and relations of the receptor to other biological factors could help in our understanding of breast cancer biology and the importance of the IGF-IR in cancer progression as well as in effective management of breast cancer.     

Pilot attempt of advanced prostate cancer treatment T3NxMx−1 by intermittent more complete androgen blockade

The aim of the prospective pilot study was evaluating efficacy and tolerance of pharma-cological more complete androgen blockade (mMAB) by using Zoladex LA 10.8 mg, Casodex 50 mg and Proscar 5 mg in patients with advanced prostate cancer (T3,Nx Mx-1). Methods: This five-year study involved 14 patients aged 67–82 years (average 73). Zoladex LA was administered subcutaneously every 3rd month of treatment, and every day 1 tablet Casodex and 1 tablet Proscar. In the time when PSA was <0.1 mg/ml Zoladex and Casodex were withdrawn, and only Proscar was left. The mMAB treatment was resumed when PSA 0.1 ng/ml. Before and every 3 months the following laboratory tests were made: PSA, sedimentation, bilirubine, transaminase, phosphatase, ultrasonography (USG); and adverse events were registered. The following criteria of assessment were adopted: CR – complete response – examination tests normal, improved condition, reduction of prostate dimension in USG and value of PSA < 0.1 ng/ml; PR – partial response i.e. no progression, the PSA level drops down to the reference values and a reduction of prostate dimension in USG occurs, NR – no response i.e. progression, increased prostate dimension in USG and/or metastases in scintygraphy, as well as PSA above normal. Results: The mean followup time was 60 months. After the initial 6 months two patients were off-therapy mMAB because they were qualified for radiotherapy. A successive patient (no. 5) was off-therapy (after 4 years) because he left Warsaw to go abroad. After 60 months results mMAB were based on 11 patients records, and a complete response was confirmed in 7 patients, partial response in 3 patients and no response in one patient. Conclusion: Results of this study show that pharmacological intermittent mMAB is an efficient way of treating advanced prostate cancer. Side effects are low and occur in moderate intensity and do not oblige to treatment withdrawal.     

Involvement of capsaicin-sensitive afferent nerves and cholecystokinin 2/gastrin receptors in gastroprotection and adaptation of gastric mucosa to Helicobacter pylori-lipopolysaccharide

Lipopolysaccharide (LPS) is one of the virulence factors in the Helicobacter pylori (Hp)-infected stomach, but it remains unknown whether single and prolonged pretreatment with Hp-LPS can affect the course of gastric damage induced by aspirin (ASA). We compared the effects of Hp-LPS with those induced by LPSs isolated from intestinal Bacteroides fragilis, Yersinia enterocolitica, and Campylobacter jejuni applied for 4 days on acute ASA-induced gastric lesions in rats. The area of ASA-induced gastric lesions, gastric blood flow (GBF), expression of mRNA and protein of leptin and plasma leptin, gastrin, interleukin-1beta, and tumor necrosis factor-alpha levels were examined. Single (once) or repeated (five times) i.p. injections of Hp-LPS (1 mg/kg) or intestinal LPSs failed to produce macroscopic gastric damage and did not affect the GBF when compared with vehicle. Hp-LPS injected repeatedly suppressed the gastric acid secretion, up-regulated leptin mRNA and protein, and increased plasma leptin and gastrin levels. Hp-LPS significantly reduced the ASA-induced gastric damage and the accompanying decline in the GBF, and these effects were significantly attenuated by capsaicin denervation and selective antagonism of cholecystokinin-B (CCK2) receptors by RPR-102681 [N-(metoxy-3 phenyl) N-(N-methyl N-phenyl-carbamylmethyl) carbamoylmethyl]-3 ureido]-3 phenyl]-2 propronique] but not by loxiglumide, an antagonist of CCK1 receptors. We conclude that 1) daily application of Hp-LPS enhances gastric mucosal resistance against ASA damage due to the increase of GBF and the expression and release of leptin and gastrin exerting trophic and gastroprotective effects, and 2) this enhanced resistance to ASA damage in Hp-LPS-adapted stomach is mediated by the sensory afferents and specific CCK2/gastrin receptors.     

Drug binding to aromatic residues in the HERG channel pore cavity as possible explanation for acquired Long QT syndrome by antiparkinsonian drug budipine

Budipine is a non-dopaminergic antiparkinsonian drug causing acquired forms of Long QT syndrome (aLQTS). As a consequence, the manufacturer has restricted the use of budipine in patients who exhibit additional risk factors for the development of Torsades-de-Pointes tachycardias (TdP). The molecular basis of this serious side effect has not been elucidated yet.Human ether-a-go-go related gene (HERG) channel block being the main cause of drug induced QT prolongation, we investigated the effect of budipine on the rapid component of the delayed-rectifier potassium current (I_K(r)) in guinea pig cardiomyocytes and on HERG potassium channels heterologously expressed in Xenopus oocytes.In guinea pig cardiomyocytes, budipine (10 M) inhibited I_K(r) by 86% but was without any effect on calcium currents. In Xenopus oocytes, HERG potassium channels were blocked by budipine with an IC₅₀ of 10.2 M. Onset of block was fast and block was only slowly and incompletely reversible upon washout.Budipine blocked HERG channels in the open and inactivated state, but not in the closed states. The half-maximal activation voltage was slightly shifted towards more negative potentials. Steady-state inactivation of HERG was also influenced by budipine. Budipine block was neither voltage- nor frequency-dependent.In HERG channel mutants Y652A and F656A, drug affinity was reduced dramatically. Therefore, these two aromatic residues in the channel pore are likely to form a main part of the binding site for budipine.In summary, this is the first study that provides a molecular basis for the budipine-associated aLQTS observed in clinical practice. Furthermore, these findings underline the importance of the aromatic residues Y652 and F656 in the binding of lipophilic drugs to HERG channels.Abbreviations I_K(r) The rapid component of the delayed-rectifier potassium current - HERG Human ether-a-go-go related gene - aLQTS Acquired Long QT syndrome - TdP Torsade-de-Pointes tachycardia E.P. Scholz and E. Zitron contributed equally to this work     

Pioglitazone,a specific ligand of peroxisome proliferator-activated receptor-gamma,accelerates gastric ulcer healing in rat

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear hormone receptor superfamily that is involved in the control of inflammation and carcinogenesis. We determined the effect of the specific PPAR-gamma ligand, pioglitazone (5-40 mg/kg intragastrically), on the healing of acetic-acid gastric ulcers in rats. At day 8 after ulcer induction, the ulcer area, the gastric blood flow and mucosal expression of proinflammatory cytokines such as interleukin-1beta, tumour necrosis factor alpha (TNF-alpha) and cyclooxygenase-1, cyclooxygenase-2, constitutive nitric oxide synthase (cNOS), inducible nitric oxide synthase (iNOS) and heat shock protein 70 (HSP70) was determined. Pioglitazone reduced the area of gastric ulcers and raised significantly the gastric blood flow at the ulcer margin and downregulated the mRNA for interleukin-1beta, TNF-alpha, cyclooxygenase-2 and iNOS while cyclooxygenase-1 mRNA was not affected. The expression of PPAR-gamma mRNA was increased in the ulcerated gastric mucosa. We conclude that pioglitazone accelerates the healing of preexisting gastric ulcers due to the hyperemia at ulcer margin and the anti-inflammatory action including suppression of interleukin-1beta, TNF-alpha, cyclooxygenase-2 and iNOS and by an overexpression of HSP70.     

Endoplasmic reticulum stress induces myostatin precursor protein and NF-kappaB in cultured human muscle fibers: relevance to inclusion body myositis

Sporadic-inclusion body myositis (s-IBM) is the most common progressive muscle disease of older persons. It leads to pronounced muscle fiber atrophy and weakness, and there is no successful treatment. We have previously shown that myostatin precursor protein (MstnPP) and myostatin (Mstn) dimer are increased in biopsied s-IBM muscle fibers, and proposed that MstnPP/Mstn increase may contribute to muscle fiber atrophy and weakness in s-IBM patients. Mstn is known to be a negative regulator of muscle fiber mass. It is synthesized as MstnPP, which undergoes posttranslational processing in the muscle fiber to produce mature, active Mstn. To explore possible mechanisms involved in Mstn abnormalities in s-IBM, in the present study we utilized primary cultures of normal human muscle fibers and experimentally modified the intracellular micro-environment to induce endoplasmic-reticulum (ER)-stress, thereby mimicking an important aspect of the s-IBM muscle fiber milieu. ER stress was induced by treating well-differentiated cultured muscle fibers with either tunicamycin or thapsigargin, both well-established ER stress inducers. Our results indicate for the first time that the ER stress significantly increased MstnPP mRNA and protein. The results also suggest that in our system ER stress activates NF-kappaB, and we suggest that MstnPP increase occurred through the ER-stress-activated NF-kappaB. We therefore propose a novel mechanism leading to the Mstn increase in s-IBM. Accordingly, interfering with pathways inducing ER stress, NF-kappaB activation or its action on the MstnPP gene promoter might prevent Mstn increase and provide a new therapeutic approach for s-IBM and, possibly, for muscle atrophy in other neuromuscular diseases.