Deinococcus as new chassis for industrial biotechnology: biology, physiology and tools.

Deinococcus spp are among the most radiation-resistant micro-organisms that have been discovered. They show remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation and oxidizing agents. Traditionally, Escherichia coli and Saccharomyces cerevisiae have been the two platforms of choice for engineering micro-organisms for biotechnological applications, because they are well understood and easy to work with. However, in recent years, researchers have begun using Deinococcus spp in biotechnologies and bioremediation due to their specific ability to grow and express novel engineered functions. More recently, the sequencing of several Deinococcus spp and comparative genomic analysis have provided new insight into the potential of this genus. Features such as the accumulation of genes encoding cell cleaning systems that eliminate organic and inorganic cell toxic components are widespread among Deinococcus spp. Other features such as the ability to degrade and metabolize sugars and polymeric sugars make Deinococcus spp. an attractive alternative for use in industrial biotechnology.

Special aspects of cosmetic spray safety evaluations: principles on inhalation risk assessment.

The consumer exposure to the vast majority of cosmetic products is limited to dermal contact. Even spray applications tend to be topically exposed to skin or hair. Besides this skin contact, spray products require additional considerations in regard to potential inhalation for building a robust and reliable safety assessment. Over the years, cosmetic industry developed prediction models for the best estimate of inhalation exposure combining data from computer simulation programs available in the market, individual real measured data and last but not least the experience from the market. Such attempt is driven by the toxicological profile of individual used ingredients. The focus of this review is on the determination of inhalation exposure, and the derivation of safe exposure levels for cosmetic spray products. Many of the methods employed to ensure product safety of cosmetic sprays in accordance with the general requirements of the EC Cosmetics Directive are based on industry experience which are not necessarily consistent across companies. This paper presents an approach to compile common principles for risk assessment and thus contribute to standardisation of safety assessment methodologies utilized for spray product evaluation without interfering with the flexibility of the individual safety assessor. It is based on the experience within the author's companies and may be useful as a support document as well for SME (Small and Medium Enterprises) companies safety assessors. In this respect it can be seen as one fundamental step in a tiered approach of cosmetic spray safety evaluation.

Mutations in fibrillin-1 cause congenital scleroderma: stiff skin syndrome.

The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence-encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor-beta (TGFbeta). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFbeta concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.

Repeated topical treatment, in contrast to single oral doses, with Vitamin A-containing preparations does not affect plasma concentrations of retinol, retinyl esters or retinoic acids in female subjects of child-bearing age.

BACKGROUND: Vitamin A is widely used in cosmetic preparations. Given that oral Vitamin A and its metabolites present a potential reproductive risk, the present study investigated the effect of topical Vitamin A on human endogenous plasma levels of Vitamin A and its metabolites. METHODS: Two groups of 14 female volunteers of child-bearing age were kept on a Vitamin A-poor diet and treated topically for 21 days with creams containing 0.30% retinol or 0.55% retinyl palmitate on approximately 3000 cm2 of their body surface area, amounting to a total of approximately 30,000 IU Vitamin A/subject/day. After a 12-day wash-out period, the study groups received single oral doses of 10,000 IU or 30,000 IU retinyl palmitate (RP), corresponding to the maximal EU allowance during pregnancy or three-times higher, respectively. Blood samples were collected over 24h on study days -3 (pre-study), 1, 21 (first and last days of topical treatment) and 34 (oral administration) at 0, 1, 2, 4, 6, 8, 12, 14-16 h and 24 h after treatment for determination of plasma concentrations of retinol (REL), retinyl palmitate (RP), oleate (RO) and stearate (RS), 9-cis-, 13-cis-, all-trans- (AT), 13-cis-4-oxo- or AT-4-oxo-retinoic acids (RAs). RESULTS: With the exception of transient mild (RP-group) to moderate (REL-group) local irritation on the treatment sites, no adverse local or systemic effects were noted. On days 1 or 21 of topical treatment, no changes were measured in individual or group mean plasma Cmax, AUC0-24 h or other pharmacokinetic parameters of REL, retinyl esters or RAs relative to pre-study data. In contrast, single oral doses of RP at 10,000 IU or 30,000 IU produced dose-related and sustained increases in Cmax and AUC0-24 h values of plasma RP, RO, RS, 13-cis- and 13-cis-4-oxo-RAs, as well as a transient increase in AT-RA. In conclusion, our results provide evidence that human topical exposure to retinol- or retinyl ester-containing cosmetic creams at 30,000 IU/day and maximal use concentrations do not affect plasma levels of retinol, retinyl esters or RAs, whereas single oral doses at 10,000 IU or 30,000 IU produce significant increases in plasma retinyl esters and RAs.

[Determination of therapy management in rectal carcinoma by staging with 18-FDG-PET]. / Bestimmung des Therapiemanagements beim Rectumcarcinom durch Staging mittels 18-FDG-PET.

The curative treatment of carcinoma of the rectum in the early stage of the disease is radical local surgery. If there is a solitary liver metastasis, resection is also a curative treatment. This report describes a female patient with rectal carcinoma, in whom a solitary liver metastasis in the left lobe was diagnosed only by FDG-PET and verified at surgery. This case report demonstrates the potential role of FDG-PET even for primary staging in detecting occult hepatic and extrahepatic metastases, thus significantly influencing the therapeutic management and prognosis of these patients.

Radiotherapy of testicular intraepithelial neoplasia (TIN): a novel treatment regimen for a rare disease.

PURPOSE: Testicular intraepithelial neoplasia (TIN) is a consistent precursor of most invasive germ cell tumors, currently treated by radiotherapy with 20 Gy, which destroys TIN but preserves Leydig cells. Nevertheless, analysis has shown dose-dependent dysfunction even with low therapeutic doses of 20 Gy in some cases. Therefore, we tested a dose reduction regimen by delivering smaller fractional doses to enhance the tolerance of Leydig cells. METHODS AND MATERIALS: Between 1993 and 1999, 9 patients were treated for TIN in a prospective multicenter trial. A total dose of 13 Gy was administered in 10 fractions of 1.3 Gy. Hormonal levels of follicle-stimulating hormone, luteinizing hormone, and testosterone were assayed serially. RESULTS: During a median follow-up time of 36 months, no patient showed evidence of local disease. A first postradiation biopsy was obtained 3-12 months after radiotherapy; 5 patients underwent a second biopsy 2-3 years after treatment. All biopsies showed a Sertoli cell-only pattern. Follicle-stimulating hormone levels continued to increase 1 year after radiotherapy, signaling eradicated spermiogenesis. Luteinizing hormone and testosterone remained within the normal range 2 years after radiotherapy. CONCLUSIONS: In the treatment of TIN, there seems to be a dose reduction potential to 13 Gy by lowering single fractional doses, which enhances the therapeutic ratio in favor of the Leydig cells.

Influence of tumor necrosis factor-alpha and silibin on the cytotoxic action of alpha-amanitin in rat hepatocyte culture.

Tumor necrosis factor-alpha is assumed to play a role in toxic liver damage. We examined whether exogenous tumor necrosis factor-alpha must be present for alpha-amanitin cytotoxicity in rat hepatocyte culture. alpha-Amanitin at a concentration of 0.1 microM, which is close to that found in intoxicated patients, inhibits RNA and protein synthesis within 12 h but cytotoxicity only occurs after a latency period and is pronounced at 36 h after the start of treatment. Tumor necrosis factor-alpha is not indispensable for the development of cytotoxicity but aggravates it and leads to a time shift towards earlier times. Lipid peroxidation is low with alpha-amanitin alone even at 36 h but markedly increased by cotreatment with tumor necrosis factor-alpha. The antioxidant silibin prevents the effect of tumor necrosis factor-alpha, indicating an involvement of reactive oxygen species. alpha-Amanitin alone does not increase but dose-dependently inhibits the expression of the antioxidant enzyme manganous superoxide dismutase and decreases the inducing effect of TNF-alpha on the expression of this enzyme. The gene expression of endogenous tumor necrosis factor-alpha in the hepatocytes is not increased but rather inhibited by alpha-amanitin treatment. The results suggest that alpha-amanitin causes delayed cytotoxicity following rapid inhibition of RNA and protein synthesis and that tumor necrosis factor-alpha shortens the latency period and aggravates the cytotoxicity by a mechanism which may involve reactive oxygen species.

Grammatical morphology and the lexicon in children with specific language impairment.

We examined the use of grammatical morphology by preschool-age English-speaking children with specific language impairment (SLI) as a function of their lexical diversity. Relative to a group of normally developing (ND) preschoolers, these children's use of finite-verb morphology logged behind expectations based on the number of different verbs they used. Noun-related morphology fell below expectations based on overall lexical diversity. Differences between the ND children and children with SLI were also seen for the slope of the increases in finite-verb morphology as a function of lexical diversity, with shallower slopes in the SLI data. The findings of this study add to existing evidence suggesting that a measure of finite grammatical-morphology use has promise as a clinical marker of SLI in English.

Impairment of TNF-alpha expression and secretion in primary rat liver cell cultures by acetaminophen treatment.

Tumor necrosis factor-alpha (TNF-alpha) is assumed to act as a mediator in toxic liver injury, aggravating the primary damage to the parenchymal liver cell, but also stimulating liver regeneration. Reports on the effect of acetaminophen in vivo on TNF-alpha transcript concentrations and serum TNF-alpha concentrations, under different experimental, or clinical conditions have yielded controversial results. We used primary rat hepatocyte and Kupffer cell cultures to test the direct action of subtoxic and toxic concentrations of acetaminophen on TNF-alpha expression and release. We observed a dose-dependent decrease of TNF-alpha mRNA in the hepatocytes, and of TNF-alpha release into the medium of hepatocyte cultures. The data also indicate an impairment of TNF-alpha release in Kupffer cell cultures after treatment with nontoxic, as well as with toxic, acetaminophen concentrations. The results suggest that inhibition of TNF-alpha expression and release in the liver is a consequence of acetaminophen exposure. It is at present unknown how this effect modulates the course of acetaminophen intoxication.

Racial factors and the risk of chronic pancreatitis.

OBJECTIVE: It is unclear why some alcohol abusers develop alcoholic cirrhosis whereas others contract chronic pancreatitis. The aim of this study was to examine the importance of race as a risk factor for the development of chronic pancreatitis. METHODS: We compared the racial status of 1883 patients discharged with a first-listed diagnosis of two diseases strongly related to alcohol abuse: 433 patients with chronic pancreatitis (ICD 5771) and 1450 patients with alcoholic cirrhosis (ICD 5712). Information came from discharge statistics maintained by two acute care hospitals in New York City and one acute care hospital in Lisbon, Portugal. The study period included the years 1989-1996 in the US and 1989-1994 in Portugal. RESULTS: A total of 215 (50%) of the 433 chronic pancreatitis patients were black compared with 333 (23%) of the 1450 patients with alcoholic cirrhosis. When adjusted for sex and hospital site, patients with pancreatitis were significantly more likely to be black than patients with cirrhosis (odds ratio 2.5, 95% confidence interval 1.9-3.2, p < 0.001). CONCLUSIONS: In comparison with white patients, black patients are two to three times more likely to be hospitalized for chronic pancreatitis than alcoholic cirrhosis. This highly significant (p < 0.001) difference was observed in both men and women: in three different hospitals, and in two different countries. The explanation is unknown, but could be related to racial differences in diet, type or quantity of alcohol consumption, smoking, or ability to detoxify substances harmful to the liver or pancreas.

Influence of acetaminophen treatment and hydrogen peroxide treatment on the release of a CINC-related protein and TNF-alpha from rat hepatocyte cultures.

Western blot analysis of conditioned media from hepatocytes exposed to H2O2 revealed that a 28 kDa protein was released dose-dependently in response to 1-10 mM H2O2. The 28 kDa protein was present in freshly isolated hepatocytes and exhibited cross-reactivity towards an antibody against CINC/gro. The intracellular amount of the protein decreased in parallel to the H2O2-induced release into the medium. The CINC-related protein was absent in media harvested after 1 h of treatment. The delivery of CINC-related protein correlated with the extent of cell damage as judged from lactate dehydrogenase leakage. Likewise, exposure of hepatocytes to 10-50 mM acetaminophen resulted in a dose-dependent release of the CINC-related protein after 24 h of culture. In contrast, monomeric CINC (molecular weight approximately 6.5 kDa) but not the 28 kDa CINC-related protein was released by lipopolysaccharide (LPS)-stimulated Kupffer cells. The amount of monomeric CINC liberated by Kupffer cells was diminished upon acetaminophen-treatment. Also, the release of tumor necrosis factor-alpha by hepatocytes was reduced after exposure to high acetaminophen doses (40-50 mM). In contrast to this finding, TNF-alpha release from hepatocyte cultures was not affected after H2O2 treatment. These data suggest that damaged hepatocytes release proinflammatory cytokines which may aggravate liver injury through activation of neutrophils and monocytes. The results indicate that the appearance of the CINC-related protein is due to impairment of plasma membrane integrity as the consequence of massive cell damage. In addition, APAP inhibited the release of monomeric CINC from LPS-activated Kupffer cells and of TNF-alpha from hepatocytes even at concentrations that were not sufficient to affect cell viability.

Enhancement of glycolysis and CO2 formation from glycerol by hydroxyl radical scavengers in rat hepatocytes.

Two agents possessing hydroxyl radical scavenger activity, dimethyl sulfoxide and tris(hydroxymethyl)aminomethane, increased the production of lactate plus pyruvate in rat hepatocytes cultured for 2 hours. Enhancement of lactate plus pyruvate formation was no longer observed in rat hepatocytes cultured for 24 hours. The formation of CO2 from glucose and from glycerol in suspensions of freshly isolated rat hepatocytes was also enhanced by the two hydroxyl radical scavengers. These results suggest that glycolysis in hepatocytes is suppressed by hydroxyl radicals formed endogenously due to oxidant stress resulting from cell isolation and that the suppression can be relieved by antioxidants.

Oxidation of Tris and formaldehyde to CO2 by neutrophil oxidants.

The buffer substance Tris is oxidized to formaldehyde by two microbicidal neutrophil oxidants, the hydroxyl radical and sodium hypochlorite. By the intact neutrophil, Tris is converted to CO2 in a process which is enhanced by stimulation with zymosan indicating that CO2 formation reflects neutrophil function. CO2 formation is more extensive at pH 6.0 than at pH 7.5 suggesting higher microbicidal activity at the low pH of inflamed tissue. In addition to Tris, its oxidation product formaldehyde is also oxidized to CO2 by hydroxyl radicals, and the formation of CO2 from formaldehyde by intact neutrophils exhibits the same features as CO2 formation from Tris. It is suggested that CO2 formation from Tris or from formaldehyde may be suitable to test for the formation of microbicidal oxidants by neutrophils.

Ortho-phenanthroline modulates enzymes of cellular energy metabolism.

The lipophilic iron chelator 1,10-phenanthroline has been used in mechanistic studies on intracellular oxidant damage because iron is assumed to play a role in the endogenous formation of highly reactive oxygen species. This study shows that 1,10-phenanthroline has enzyme-modulatory properties in addition to its antioxidant activity. In rat hepatocytes, 1,10-phenanthroline caused inhibition of respiration and enhancement of cellular ATP content, pyruvate release and CO2 formation from glycerol resulting from a modulatory action of 1,10-phenanthroline on various enzymes involved in cellular energy metabolism. In intact mitochondria and in submitochondrial particles, oxygen consumption, complex I activity, and ATPase degradation are inhibited by 1,10-phenanthroline. In submitochondrial particles, complex II activity can also be suppressed by 1,10-phenanthroline. The purified cytosolic enzymes lactate dehydrogenase and glycerol-3-phosphate dehydrogenase are inhibited while purified glyceraldehyde-3-phosphate dehydrogenase is activated by 1,10-phenanthroline. The results suggest that 1,10-phenanthroline modulates various enzyme activities linked to cellular energy metabolism and that this property must be taken into account when using 1,10-phenanthroline as a tool in experiments on oxidant effects in cells.

Human immunodeficiency virus-related microvasculopathy and Kaposi's sarcoma: a case-control study.

Ocular microangiopathic syndrome including retinal and conjunctival abnormalities is frequently found in patients with human immunodeficiency virus type 1 (HIV-1) disease. Kaposi's sarcoma (KS) is the most frequent neoplasia found in patients with HIV-1 disease. We have recently reported a significant association between conjunctival microvasculopathy and KS in 117 patients with HIV-1 disease. The objective of the present study was to determine whether this association is existent when matched patients with and without KS are compared. A total of 22 matched pairs were obtained under consideration of the absolute CD4+ lymphocyte count, Walter Reed (WR) classification, gender, and serum levels of beta-2-microglobulin and neopterin. Conjunctival microangiopathy was determined for each eye by a standardized rating scale ranging from 0 to 5, allowing a reliable and valid quantification of conjunctival blood-flow sludging. The mean value obtained for conjunctival sludge was 1.8 (SEM, 0.4) for patients without KS and 3.2 (SEM, 0.3) for patients with KS, demonstrating a clinically and statistically significant difference between the two groups (Student's t = 3.0; P = 0.003). This difference was higher when patients with a CD4+ lymphocyte count exceeding 200/microliters were regarded. Similar factors or mechanisms may contribute to HIV-related conjunctival microvasculopathy and KS.

Urodynamic and clinical aspects of ileal low pressure bladder substitutes.

Twenty-three patients with an ileal bladder substitute formed after cystectomy for invasive bladder cancer were evaluated clinically and urodynamically between 3 and 38 months post-operatively. The urodynamic measurements were compared with the clinical findings. After re-education of the patients' voiding habits the mean voiding volumes of the bladder substitutes stabilised 6-9 months post-operatively at 350 ml. The frequency of micturition was 3 to 5 times during the day and once or twice at night. The maximum functional capacity (maximum voiding volume) was about 490 ml. Ninety-one per cent of the patients were continent during the day 18 months after the operation and 82% were continent during the night. Micturition was problem-free with an average maximum flow of 25 ml/s and an average micturition time of 50 s. The mean voiding volume of ileal bladder substitutes was 50% of the measured cystometric capacity; the maximum functional capacity (= max. micturition volume) was 80% of the cystometric capacity. The average basal pressure was < 20 cm H2O from the third post-operative month onwards. Eleven of the 23 patients had contractions in the bladder substitute (average at 30 cm H2O) at 55-76% of the maximum cystometric capacity or at approximately 90% of the maximum functional capacity. Such spike waves had no clinical or radiological consequences. If the patients were shown how to increase the functional capacity of a reservoir made from only 40 cm of ileum, the clinical results were excellent.

Late results in patients treated with pi-mesons for bladder cancer.

BACKGROUND: The treatment of bladder cancer with 60 converging pion beams was expected to have certain dose-distribution advantages with possibly fewer side effects than other high linear-energy-transfer (LET) radiation therapies, such as neutrons. RESULTS: Early results were promising: 20 of 24 (83%) evaluable patients with sessile invasive bladder carcinomas had clinically complete responses. However, only 3 of 10 (30%) evaluable patients with superficial bladder tumors had clinically complete responses. This article reports the long-term follow-up (6-8 years) of these patients with emphasis on the late side effects of pion radiation therapy. Thirty-eight of the 41 (93%) patients treated died after a median survival time of 17 months (range, 4-98 months). Seventeen (45%) died of metastatic disease (in two instances, this was combined with a local recurrence) 5-27 months after radiation therapy. Four (10%) died of locally progressive disease, and eight (21%) died of late side effects of radiation therapy 9-98 months after treatment. All these patients were treated with more than 33 pion Gy and had generally a symptom-free interval of 9-18 months. The observed side effects were severe, consisting of chronic inflammation and vascular damage in the pelvic region often followed by ulceration, fistulas, and perforations throughout the intestines. In 11 patients, cystectomy and urinary diversion was necessary because of excessive fibrosis and bladder shrinkage. In eight patients, a colostomy was required for stenotic inflammatory disease, necrosis, and perforations of the intestines. The remaining nine patients (24%) died of causes unrelated to the primary disease 4-60 months after radiation therapy. CONCLUSIONS: The results of the first Phase I/II trial using the Swiss piotron showed a high complete response rate in patients with sessile bladder cancers but also a high incidence of local recurrences and severe, in some instances lethal side effects. Although it is expected that these results will be the basis for future improvements, particularly regarding dosing and fractionation, this experience emphasizes the need for a sufficiently long observation period before reaching conclusions about any high LET treatment, such as neutron, pion, or heavy ion radiation therapy.

[Pamidronate in the treatment of tumor-associated hypercalcemia]. / Pamidronat in der Behandlung der tumorassoziierten Hypercalcämie.

After a 48-hour rehydration period 28 of 31 patients with cancer-associated hypercalcemia (serum calcium greater than or equal to 2.8 mmol/l) were treated intravenously with the bisphosphonate pamidronate. In three patients fluid repletion with 0.9% saline solution had already normalized serum calcium levels. Pamidronate was given in a single infusion on day 0, the dose of pamidronate adapted to the severity of hypercalcemia. If the serum calcium concentration was greater than or equal to 2.8 mmol/l on day 3, application of pamidronate was repeated. In all patients normocalcemia was restored; mean serum calcium decreased from 3.2 +/- 0.35 on day 0 to 2.15 +/- 0.32 on day 12. Hypercalcemia recurred in 11 patients, seven of these underwent pamidronate treatment according to the same therapeutical regimen. Normal calcium levels were attained in five cases. Side effects were of minor gravity: brief hyperthermia occurred in four patients and transient, asymptomatic hypocalcemia was noticed in nine cases.

Ileal bladder substitute: antireflux nipple or afferent tubular segment?

Spheroidal bladder substitutes made from double-folded ileal segments, similar to Goodwin's cup-patch technique, are devoid of major coordinated wall contractions. This, together with the reservoir's direct anastomosis to the membranous urethra, prevents major intraluminal pressure peaks and assures a residue-free voiding of sterile urine. In order to determine whether, under these conditions, an afferent tubular isoperistaltic ileal segment of 20-cm length protects the upper urinary tract as efficiently as an antireflux nipple, 60 male patients who were subjected to radical cystectomy were prospectively randomised to groups in which a bladder substitute was formed together with either of these 2 antireflux devices. An analysis of the results obtained in 20 patients from each group who could be followed for more than 1 year (median observation time 30 and 36 months) showed no differences between the groups in metabolic disturbances, kidney size, reservoir capacity, diurnal and nocturnal urinary continence, the incidence of urinary tract infection or episodes of acute pyelonephritis. Later than 1 year postoperatively, intravenous urograms of the renoureteral units of 25% of the patients with antireflux nipples showed persistent but generally slight dilatation of the upper urinary tracts. This observation was significantly more frequent than it was in patients with afferent tubular segments. Urodynamic and radiographic studies showed that the competence of the antireflux nipples was secured by the raised surrounding intravesical pressure. This, however, also resulted in a transient functional obstruction, and a gradual rise of the basal pressure in the upper urinary tracts was recorded. In patients with afferent ileal tubular segments, contrast medium could be forced upwards into the renal pelvis when the bladder substitutes were overfilled. However, despite raised intravesical pressures, peristalsis in the isoperistaltic afferent tubular segment gradually returned contrast medium back to the reservoir. Our results suggest that the combination of an ileal low-pressure reservoir together with an afferent tubular isoperistaltic limb is at least as good as an antireflux nipple valve. Moreover, the use of the afferent ileal limb makes it possible to resect the distal and often diseased ureters together with the paraureteric lymphatics at a safe distance from the bladder tumor. This avoids also distal ischemic ureteric stenosis and makes possible a simple end-to-side ureterointestinal anastomosis with a small complication rate.