[Quantitative analysis of corneal subbasal nerve plexus with in vivo confocal laser scanning microscopy]. / Quantitative Analyse des subbasalen Nervenplexus der Kornea mittels in vivo konfokaler Laser-Scanning-Mikroskopie.

BACKGROUND: An analysis of the corneal subbasal nerve plexus (SNP) allows an evaluation of the peripheral neuropathy in cases of degenerative diseases. In order to study the SNP structures quantitatively the automatically calculated morphological and topological parameters are required. METHODS: In vivo confocal laser scanning microscopy (Heidelberg Retina Tomograph II/Rostock Cornea Module) was performed in healthy volunteers as well as patients with severe diabetic neuropathy. An adapted image processing algorithm was used to preprocess, segment and evaluate quantitatively the nerve fibers of the SNP. Data sets were analysed statistically. RESULTS: The developed algorithm allows an automated detection of SNP structures. Furthermore, it allows the collection of data based on morphological and topological parameters. The main parameters that show significant differences between healthy cornea and cases of diabetic neuropathy are nerve fibre density and length, number of branching, tortuosity and number of terminal and crossing points. All parameters of the measurements can be used isolated, combined or weighted for quantification of the SNP networks. CONCLUSION: The presented fully automated preprocessing eliminates a large number of motion-induced artefacts. The quality of the resulting pictures allows an automated quantification using characteristic measurements. This represents an in vivo, non-invasive technology analysing degenerative changes of SNP especially in the course of diabetes mellitus.

Apoptosis induced by proteasome inhibition in cancer cells: predominant role of the p53/PUMA pathway.

The proteasome has emerged as a novel target for antineoplastic treatment of hematological malignancies and solid tumors, including those of the central nervous system. To identify cell death pathways activated in response to inhibition of the proteasome system in cancer cells, we treated human SH-SY5Y neuroblastoma cells with the selective proteasome inhibitor (PI) epoxomicin (Epoxo). Prolonged exposure to Epoxo was associated with increased levels of poly-ubiquitinylated proteins and p53, release of cytochrome c from the mitochondria, and activation of caspases. Analysis of global gene expression using high-density oligonucleotide microarrays revealed that Epoxo triggered transcriptional activation of the two Bcl-2-homology domain-3-only (BH3-only) genes p53 upregulated modulator of apoptosis (PUMA) and Bim. Subsequent studies in PUMA- and Bim-deficient cells indicated that Epoxo-induced caspase activation and apoptosis was predominantly PUMA-dependent. Further characterization of the transcriptional response to Epoxo in HCT116 human colon cancer cells demonstrated that PUMA induction was p53-dependent; with deficiency in either p53 or PUMA significantly protected HCT116 cells against Epoxo-induced apoptosis. Our data suggest that p53 activation and the transcriptional induction of its target gene PUMA play an important role in the sensitivity of cancer cells to apoptosis induced by proteasome inhibition, and imply that antineoplastic therapies with PIs might be especially useful in cancers with functional p53.

Efficacy and safety of a new ready-to-use recombinant human growth hormone solution.

We report 24-month interim results of two multicenter phase III studies in previously untreated children with growth failure secondary to GH deficiency (GHD) that were paramount to the development of a new recombinant human GH (rh- GH, somatropin), approved as the first 'biosimilar' in Europe. Study 1 consisted of 3 parts performed in 89 children. The objective was to compare efficacy and safety of the lyophilized formulation of the new somatropin [Somatropin Powder (Sandoz)] with a licensed reference rhGH preparation and the liquid formulation of the new somatropin [Somatropin Solution (Sandoz)] and to assess long-term efficacy and safety of this ready-to-use Somatropin Solution. Study 2 was performed in 51 children and designed to demonstrate efficacy and safety of Somatropin Powder and to confirm its low immunogenic potential; rhGH was given sc at a daily dose of 0.03 mg/kg. Primary [body height, height SD score (HSDS), height velocity, and height velocity (HV) SD score (HVSDS)] and secondary [IGF-I and IGF binding protein 3 (IGFBP-3)] efficacy endpoints and safety parameters were assessed regularly. In study 1, all treatments showed comparable increases in growth. The baseline-adjusted difference between Somatropin Powder and the reference rhGH product in mean HV was -0.20 cm/yr (95% confidence interval (CI) [-1.34;0.94]) and in mean HVSDS was 0.76 (95% CI [-0.57;2.10]) after 9 months. These very small differences demonstrate comparable therapeutic efficacy between the two treatments. The results of study 2 were consistent with those seen in study 1. Equivalent therapeutic efficacy and clinical comparability in terms of safety and immunogenicity between Somatropin Powder and the reference rhGH product and between Somatropin Powder and Somatropin Solution was demonstrated. The safety and immunogenicity profiles were similar and as expected from experience with rhGH preparations.

[Knowledge and use of Pap-smear among HIV-positive women]. / Importanza della conoscenza del Pap-test e suo utilizzo tra le donne HIV-positive.

AIM: HIV-positive women are at increased risk for preneoplastic lesions and invasive cervical cancer (ICC). The occurrence of these lesions can be substantially reduced by appropriate cervico-vaginal screening protocols (i.e., Pap-test). The aim of study was to assess: 1) awareness of Pap-smear and 2) the association between awareness of Pap-smear and screening attitudes of HIV-positive women. METHODS: Three-hundred and ninety HIV-positive women who attended the HIV outpatient gynecological unit of the National Institute for Infectious Diseases, Rome, from January 2003 to April 2005 were included in this investigation. These 390 women were interviewed to assess whether they were aware that Pap-test was a preventive tool against cervical cancer. In addition, past history of Pap-test, socioeconomic condition, history of HIV infection, and sexual habits were investigated. Odds ratios (OR) and 95% confidence intervals (CI) were used to assess the association between knowledge of Pap-test and covariates. RESULTS: Of these 390 HIV-positive women, 54.6% were not aware that Pap-test could prevent ICC. Women with a low educational level (OR = 6.6) or women who originated from Africa (OR = 6.5) were more likely to be unaware of Pap-test. Lack of Pap-test awareness was strongly associated with negative history for lifetime Pap-test (OR = 4.7). CONCLUSIONS: We showed that a large proportion of HIV-infected women are not aware that ICC could be prevented through Pap-test screening, and that lack of Pap-test screening is strongly associated with lack of awareness. The need for Pap-test counseling targeted to HIV-infected women clearly emerges from our findings.

A survey of transformation markers in differentiating epidermal cell lines in culture.

Primary mouse epidermal cells underwent spontaneous malignant transformation in culture. TWelve malignant epidermal cell lines were established which produced squamous cell carcinomas in syngeneic hosts. These lines were used to define criteria for recognizing transformed epidermal cells in vitro. Growth in suspension in agar, agarose, or Methocel was minimal for 11 of the lines. All lines tested retained specific epidermal antigens (pemphigus, pemphigoid, keratin) by indirect immunofluorescence, but keratin content was reduced when quantified by radioimmunoassay. Basal activity of ornithine decarboxylase and activity induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate were variable among lines. All malignant lines as well as normal epidermal cells grew well at reduced extracellular calcium concentrations. When the extracellular calcium was elevated, normal cells ceased proliferation, terminally differentiated, and sloughed from the culture dish, while malignant cells continued to proliferate although they expressed differentiative functions. These results indicate that malignant transformation in epidermis is associated with a fundamental alteration in the program of terminal differentiation which allows some cells to escape the proliferative block and cell death which accompanies differentiation in normal keratinocytes. This alteration should be useful to select for transformants during the process of carcinogenesis in vitro.

Recombinant human interleukin 4 has antiproliferative activity on human tumor cell lines derived from epithelial and nonepithelial histologies.

Interleukin 4, a T cell-derived 20-kDa glycoprotein, plays an important role in regulating the immune response of B cells, T cells, and macrophages against infections and malignant cells. For this reason recombinant human interleukin 4 (rhIL-4) has entered early clinical trials in cancer patients. In the present study we report that rhIL-4 has an antiproliferative effect on five of nine cell lines derived from human colon tumors, head and neck tumors, and glioblastomas as measured by a decrease of colony formation in human tumor cloning assays. All of the cell lines with in vitro responsiveness express at least 100 high-affinity receptors for human interleukin 4 per cell on their cell surface, whereas the nonresponsive tumor cell lines lack expression of high-affinity receptors for human interleukin 4 on their cell surface. In the next series of experiments we have xenotransplanted some of the responsive cell lines into athymic nude mice. Subsequently, the animals were treated s.c. twice daily with 0.5 mg/m2 rhIL-4 or control vehicle for at least 12 days. There was a clear growth inhibition of these xenotransplanted tumors in the mice treated with rhIL-4. Histology of the tumors in both groups revealed no marked infiltration with murine hematopoietic and lymphocytic cells as evaluated by staining with a rat anti-mouse CD45 antibody. We conclude that rhIL-4 has a direct therapeutic activity on the growth of some human epithelial and nonepithelial tumor cell lines which, along with its regulatory function on hematolymphopoietic cells, makes this cytokine an interesting candidate for experimental tumor therapy.

Valsartan versus ACE inhibition after bare metal stent implantation--results of the VALVACE trial.

The use of ACE inhibitors (ACE-i) represents an Ia recommendation in the treatment of patients with STEMI and NSTEMI. However, results of smaller studies suggest an increase of in-stent-restenosis under ACE-i administration. The effects of ACE-i and valsartan after bare metal stent implantation of the culprit type B2/C lesion should be compared. Seven hundred patients were treated either by ACE-i in cases of LVEF<50% or 80 mg valsartan in cases of LVEF> or =50%. Restenosis rates after 6 months were analysed in 399 patients under valsartan and 224 patients under ACE-i with control angiography and major adverse cardiac events (death, infarction, reintervention) in a follow-up of up to 4 (mean 2.6) years in all patients. In-stent-restenosis was found in 19.5% under valsartan and in 34% under ACE-i (p<0.005). In diabetic patients, restenosis occurred in 24% under valsartan and in 43% under ACE-i (p<0.01). In initial acute coronary syndrome (ACS), restenosis rate was 14% under valsartan and 43% under ACE-i (p<0.0001). In stable angina, restenosis rates were 26.5% and 27.5%, respectively. Total MACE rates revealed significant differences in ACS due to reintervention rates of 22% and 7% under ACE-i and valsartan (p<0.0001). The administration of 80 mg valsartan after bare metal stent implantation leads to a reduction of in-stent-restenosis compared to ACE-i. This effect is mainly due to beneficial effects of valsartan in cases with initial ACS. Major differences between ACE-i and valsartan are discussed including inflammation, activation of neutrophils, mode of bradykinin activation, AT2 receptor stimulation and apoptosis of smooth muscle cells.

Clonal growth of mouse epidermal cells in medium with reduced calcium concentration.

Mouse keratinocytes can be grown at clonal densities in dermal fibroblast conditioned medium with a calcium concentration of 0.02 MM. Colony forming efficiencies of approximately 1-3% can be achieved with primary and secondary cultures and up to 6% with selected subclones. A substrate of frozen-thawed dermal fibroblasts enhances colony formation over plastic. Colony size increases more rapidly in the presence of epidermal growth factor in conditioned medium. Conditioning of medium by fibroblasts is optimum after day 6 of culture and conditioned medium can be sotred at -20 degrees C for at least 4 mo.

The bimodal mortality pattern of systemic lupus erythematosus.

The changing pattern of mortality in systemic lupus erythematosus (SLE) led to an examination of the deaths in a long-term systematic analysis of 81 patients followed for five years at the University of Toronto Rheumatic Disease Unit. During the follow-up 11 patients died; six patients died within the first year after diagnosis (group I) and five patients died an average of 8.6 years (from 2.5 to 19.5 years) after diagnosis (group II). In those who died early, the SLE was active clinically and serologically, and nephritis was present in four. Their mean prednisone dose was 53.3 mg/day. In four patients a major septic episode contributed to their death. In those who died late in the course of the disease, only one patient had active lupus and none had active lupus nephritis. Their mean prednisone dose was 10.1 mg/day taken for a mean of 7.2 years. In none was sepsis a contributing factor to their death. All five of these patients had had a recent myocardial infarction at the time of death; in four, ti was the primary cause of death. Mortality in SLE follows a bimodal pattern. Patients who die early in the course of their disease, die with active lupus, receive large doses of steroids and have a remarkable incidence of infection. In those who die late in the course of the disease, death is associated with inactive lupus, long duration of steroid therapy and a striking incidence of myocardial infarction due to atherosclerotic heart disease.

Cyclopent[a]anthraquinones as DNA intercalating agents with covalent bond formation potential: synthesis and biological activity.

A series of mitomycin C (MMC) analogues, namely cyclopentanthraquinone derivatives, were synthesized via Diels-Alder cyclization of naphthoquinone with 1-vinylcyclopent-1-enes. These new compounds are planar structures, like MMC, and bear an aziridine ring and a methyl carbamate side chain. After bioreduction, they are anticipated to be capable of intercalating into double-stranded DNA and bind covalently. Structure-activity relationships were studied. Of these compounds, 2,3-aziridino-4-[[(methylamino)carbonyl]methyl] cyclopent[alpha]anthracene-6,11-dione (4) was shown to have inhibitory activity against several leukemic and solid tumor cell lines. Mice (BDF1) bearing Lewis lung adenocarcinoma were treated with 4 and MMC (i.p., QD x 5). At a dose of 30.0 mg/kg, compound 4 was as effective as MMC (0.8 mg/kg). Compound 4 appears to be less toxic than MMC. DNA unwinding assay indicated that 4 is able to intercalate into DNA double strands and is also a topoisomerase II inhibitor.

Antiproliferative effect of human interleukin-4 in human cancer cell lines: studies on the mechanism.

Interleukin-4 (IL-4) plays an important role in activating the immune system against malignant cells. The human interleukin-4 receptor (hIL-4R) is not only expressed by hematopoietic cells but also on a large number of tissue specimens which include colon, breast and lung carcinomas. In this study we report that rhIL-4 has an antiproliferative effect on 2 out of 3 non-small cell lung carcinoma (NSCLC) cell lines in vitro as measured by human tumor cloning assays (HTCA). In comparison, rhIL-4 had no effect on the growth of small cell lung carcinoma cell lines (SCLC) in vitro. The response towards the cytokine is correlated with expression of at least 1500 high affinity receptors/cell for hIL-4 on the responsive cell lines. Xenotransplanting the human lung tumor cell lines into nude mice followed by 12 days of systemic treatment of the mice with rhIL-4 revealed a significant growth retardation of the IL-4R positive NSCLC cell lines when compared with the controls, whereas the growth of the IL-4R negative SCLC cell lines was unaffected also in vivo. Studies of possible mechanisms involved in the antiproliferative effect of rhIL-4 showed that rhIL-4 does not induce apoptosis or modulation of the transcription factor c myc in the responsive NSCLC cell lines. Additionally, the expression of the epidermal growth factor receptor (EGFR), which is discussed as mediating autocrine/paracrine growth stimulation of NSCLC, is unaffected by rhIL-4. However, we have observed that rhIL-4 inhibited G1-S-phase cell cycle progression. We conclude that rhIL-4 has an antiproliferative effect on the growth of some NSCLC in vitro and in vivo. The mechanisms involved remain to be further elucidated.

Effects of recombinant growth hormone therapy on thyroid hormone concentrations.

BACKGROUND AND OBJECTIVE: There are numerous, often contradictory reports on the effects of growth hormone (GH) therapy on thyroid function. The aim of this study was to assess the effect of such therapy on serum concentrations of thyroid hormones in GH-deficient children euthyroid prior to the treatment, and to determine the necessity of thyroid hormone administration in these patients. MATERIAL AND METHODS: The study included 32 GH-deficient patients in the first stage of sexual development, in whom disorders of thyroid function could be excluded. The inclusion criteria were based on clinical examination and levels of thyroxine (T4), triiodothyronine (T3), free thyroxine (fT4), free triiodothyronine (fT3), reverse triiodothyronine (rT3), thyrotropin (TSH) before and after stimulation with thyrotropin-releasing hormone (TRH). Recombinant growth hormone (rGH) (Genotropin 16U, Pharmacia) was administered at a dose of 0.7 U/kg/week. Fasting blood samples were drawn before treatment and after 3, 6, 9 and 12 months of therapy. Thyroid hormones were measured using RIA and IRMA methods. RESULTS: There were no physical signs of hypothyroidism in the patients examined during 12 months of rGH administration, and the satisfactory growth rate was achieved. T4 levels decreased in the first 3 months but remained within the normal range, and then returned to the values prior to the treatment. A similar trend was observed for fF4, with 28.5% of patients exhibiting fF4 levels below the normal in the 3rd month. An increase during the first 3 months of therapy was observed in the cases of T3 (statistically non-significant) and fT3, and these values then fell to levels within the normal range of patients' age. During treatment, TSH levels decreased but remained within the normal range. CONCLUSIONS: A transient decrease in T4 concentrations in the 3rd month with unchanged T3 and an increase in fT3 concentrations probably result from the effect of rGH on the peripheral metabolism of thyroid hormones. The results obtained do not support the use of thyroid hormone therapy with levothyroxine during the first year of rGH therapy in patients who are initially euthyroid.

The behaviour of some enzymes in the mouse liver due to chronic benzene intoxication.

1. Long trem benzene action brings about a permanent decrease in oxidoreductive enzymes and active transport as well as an inactivation of the lysosomal apparatus in liver-cells. 2. Particular liver lobule zones show different sensitivity to toxic action of benzene which might result from their different role in the processes of benzene biotransformation.

Histochemical and histoenzymatic changes in mouse liver in subacute benzene intoxication.

The investigations were performed on mice. They were divided into a control group and 4 experimental groups. The experimental animals were administered intraperitoneally benzene 6 X every 24 h. The animals were decapitated 30 min. 4, 12 and 24 h after the last benzene administration. During the experiment, dyeing for neutral lipids and glycogen was carried out, and the activity of NADH2-r.t., SDH, G-6-Pase, ATP-ase and ACP was estimated. A decrease of glycogen content in liver cells, deviations in the amount of neutral lipids, reversible decrease of mitochondrial enzymes activity, and intensification of the processes of intracellular catabolism were found.

Energy requirement for eating in cattle.

We determined the energy requirement for the activity associated with eating in an experiment with four adult Hinterwälder steers weighing 640+/-25 kg BW (mean+/-SD), using indirect calorimetry in respiration chambers. Heat production was measured during 3 h with and 3 h without ad libitum access to wheat straw, and the energy cost of eating was calculated as the difference, which was on average 27+/-13 J/(min x kg BW) (mean+/-SD). Straw intake, the time spent for eating, and the number of jaw movements were recorded. Values for the energy cost of eating reported in the literature are similar to those in this study, and values for ruminating amount to approximately 27% of those for eating. The energy cost of eating (literature values) varies greatly between feeds and feed treatments, when related to the amount of feed ingested, but it was relatively constant when related to time spent eating and was similar for cattle, sheep, and horses, when related to BW or metabolic BW (35 J/[min x kg BW] or 124 J/ [min x kg BW.75]). Calculations indicated that in high-quality roughage 10% of the ME and in untreated straw approximately 30% of the ME provided by the feed are used for eating and ruminating. This might be the main reason for the lower efficiency of ME utilization in roughages. The energy requirement for eating and ruminating should therefore be considered as a distinct proportion of the total ME requirement.

[Congenital adrenal hyperplasia conditioned by 21beta-hydroxylase deficiency - clinical considerations]. / Wrodzony przerost kory nadnerczy uwarunkowany niedoborem 21beta-hydroksylazy - rozwazania kliniczne.

The authors of the paper present three cases of nonclassical forms of congenital adrenal hyperplasia conditioned by 21 beta-hydroxylase deficiency occuring within the same family. They point out significant difficulties related to the detection of such anomalies, and they analyse in detail clinical symptoms occuring in this disease entity.

[Aldolase activity and the lactate level in the cord blood as objective indicators of the status of newborn infants by the Apgar score]. / Wartosci aktywnosci aldolazy i poziomu mlecznów w krwi pepowinowej jako obiektywizacja oceny stanu noworodków wg skali Apgar.

In 63 newborns with general clinical status evaluated by means of the Apgar score the lactate level and aldolase activity were determined in cord blood. A greatly raised mean level of both these biochemical indicators of tissue hypoxia was demonstrated in newborns with low Apgar score. A considerable scatter of the determined biochemical parameters was noted in newborns born in serious condition and having identical low Apgar score.

[Evaluation of glucose and cholesterol levels in the mother and newborn infant in the perinatal period]. / Ocena poziomu glukozy i cholesterolu u matki i noworodka w okresie okoloporodowym.

In 30 live born newborns the connections were assessed between glucose and cholesterol levels in umbilical cord blood after birth, and also in maternal blood before and two days after birth. Statistically significant differences of glucose and cholesterol levels were found between mothers and newborns in the perinatal period. Attention is called to greater independence of cholesterol (total, free and esterified) levels in newborn and mother in perinatal period.

[Effect of marked obesity on the emotional status of children]. / Wplyw otylosci znacznego stopnia na stan emocjonalny dziecka.

Fifty children with overweight exceeding 50% of the expected weight were studied for establishing the occurrence of emotional disturbances caused by lack of acceptance by peers. Obese children were found to suffer often from discrimination by their schoolmates which was, in some degree, connected with their low fitness. This may lead to various emotional disturbances requiring even psychotherapeutic measures.

HLA antigens in osteitis condensans ilii and ankylosing spondylitis.

Using a standard microdroplet lymphocyte cytotoxicity test for tissue typing, the distribution of the HLA antigens was determined in 37 female patients; 25 with osteitis condensans ilii (OCI) and 12 with ankylosing spondylitis (AS). Although low back pain was a common feature of OCI, none of these patients exhibited the limitation of spinal involvement, radiological evidence of spondylitis, or progressive clinical course seen in the AS group. Four of the 25 patients with OCI (16 per cent) were B27 positive vs 11 of the 12 patients with AS (92 per cent). These results suggest that OCI is not a variant of AS in women.