Histomorphometric and oxidative evaluation of the offspring's testis from type 2 diabetic female rats treated with metformin and pentoxifylline.

Type 2 diabetes mellitus (T2D) during pregnancy is characterized by high levels of reactive oxygen species and pro-inflammatory factors in the placenta. Once these reactive species reach the foetus, they trigger physiological adaptations that allow the foetus to survive, but programme the organism to develop metabolic disorders in adulthood. The male reproductive system is highly susceptible to foetal programming. This study aimed to investigate the effects of intrauterine exposure to T2D on testicular histomorphometry and redox homeostasis of adult rats and evaluate the effects of maternal treatment with metformin and pentoxifylline. Female rats were induced to T2D, then treated with metformin and pentoxifylline, or co-treated with both drugs. The females were mated, the male offspring were sacrificed on postnatal day 90, and the testicles were collected for analysis. Metformin protected the tubular compartment, with the maintenance of the Sertoli cell population and daily sperm production. Pentoxifylline attenuated the effects of diabetes on Leydig cells, in addition to stimulating testosterone production and lowering lipid peroxidation. Intrauterine exposure to T2D results in important testicular alterations that compromise gonadal function, and the co-treatment with metformin and pentoxifylline may represent a promising therapy that attenuates these effects by combining the positive influences in both the tubular and interstitial compartments of the testicular parenchyma.

High doses of eugenol cause structural and functional damage to the rat liver.

Eugenol is a phenolic compound found in clove extract and extensively used in traditional medicine. It is unclear whether its intake can cause positive or negative effects on liver morphology and physiology in healthy individuals. Thus, we aimed to evaluate liver parameters of rats treated with 10, 20, and 40 mg kg-1 eugenol. After 60 days of treatment, liver samples were collected and analyzed by biometric, histological, biochemical, and oxidative analyses. Our results showed that 10, 20, and 40 mg kg-1 eugenol did not alter body and liver weights, serum and hepatic ALT levels and catalase, glutathione-s-transferase, total, Ca2+, and Mg2+ ATPases activities in treated animals. However, 20 and 40 mg kg-1 eugenol reduced Na+/K+ ATPase pump activity and blood glucose levels. They also increased hepatic glycogen content, superoxide dismutase activity, ferric reducing antioxidant power, and nitric oxide and malondialdehyde levels. Still, 20 and 40 mg kg-1 eugenol caused structural and functional damage to the liver tissue of eugenol-treated rats. We concluded that 10 mg kg-1 eugenol is a safe dose for consumption in long-term treatment for rats. Doses higher than 20 mg kg-1 lead to hepatic damage that can impair vital processes of liver functionality.

Different Routes of Administration Lead to Different Oxidative Damage and Tissue Disorganization Levels on the Subacute Cadmium Toxicity in the Liver.

The toxic effects of cadmium (Cd) on hepatic parameters are widely described in the literature. Experimental models often make use of the intraperitoneal route (i.p.) because it is easier to apply, while in the oral route, Cd poisoning in humans is best represented by allowing the metal to pass through the digestive system and be absorbed into the bloodstream. Thus, this study investigated the Cd exposure impact on the liver, by comparing both i.p. and oral routes, both in single dose, in addition to the oral route in fractional doses. Swiss adult male mice received CdCl2 1.5 mg/kg i.p., 30 mg/kg oral single dose, and 4.28 mg/kg oral route in fractional doses for 7 consecutive days. Cd bioaccumulation was observed in all animals exposed to Cd. Hepatic concentrations of Ca and Fe increased only in the fractionated oral route. Liver activities of SOD and CAT increased only by oral single dose. GST decreased in all forms of oral administration, while MDA decreased only in i.p. route. Liver weight and HSI increased in the i.p. route, while organ volume increased in all forms of oral administration, and liver density increased in all animals exposed to Cd. In hepatic histomorphometry, the changes were more evident in oral administration, mainly in exposure to metal in a single dose. Thus, the subacute administration of Cd in different routes of administration leads to different changes in liver poisoning.

Neonatal meningitis, endocarditis, and pneumonitis due to Streptococcus gallolyticus subsp. pasteurianus: a case report.

BACKGROUND: Streptococcus pasteurianus is a rare cause of neonatal infection, with only 3 cases reported in the USA and 18 cases reported in other countries within the past decade. Neonatal S. pasteurianus infection typically presents as meningitis. This case report describes the first neonatal case of S. pasteurianus endocarditis in the literature, in addition to a neonatal case of S. pasteurianus infection presenting as pneumonitis without meningitis. The S. pasteurianus infections in these two cases are unusual not only because of how rare this particular pathogen is, but also because of the atypical clinical manifestations. CASE PRESENTATION: The first patient is a full-term male infant admitted to NICU at 20 h of life due to respiratory distress. He was empirically started on ampicillin and gentamicin for presumed sepsis. Laboratory analysis of cerebral spinal fluid obtained after initiation of antibiotics was suggestive of partially treated meningitis. Blood cultures came back positive for S. pasteurianus. The neonate was transitioned from ampicillin to cefepime, while gentamicin was continued. Echocardiograph showed a possible tricuspid valve vegetation concerning for endocarditis. Due to the unusual complication of endocarditis, the patient remained on IV cefepime for 28 days rather than the more conventional duration of 14-21 days reported in the literature. The baby clinically improved with no evidence of thrombi or vegetations on repeat cardiac echo. The second patient is a full-term male infant who required intubation at birth for respiratory distress. Chest X-ray findings were concerning for meconium aspiration with pneumonitis. The baby went into septic shock and was empirically started on ampicillin and gentamicin. Blood cultures came back positive for S. pasteurianus, while cerebral spinal fluid and urine cultures were negative. Ampicillin and gentamicin were discontinued after 3 days and the baby was started on cefepime and clindamycin for a total 14-day course. The baby clinically recovered and was discharged from NICU without any sequelae. CONCLUSIONS: These two cases highlight the importance of recognizing S. pasteurianus as a potential cause of neonatal sepsis and the importance of recognizing endocarditis and pneumonitis as possible clinical manifestations of this infection.

Recurrent Kawasaki Disease: A Case Report of Three Separate Episodes at >4-Year Intervals.

Kawasaki disease (KD) is a self-limited systemic vasculitis, most often occurring in children 1⁻5 years old. It has a 2% recurrence rate and is associated with coronary aneurysms (CA), which can develop within two weeks of onset. A 25% increased risk is noted in patients who are recalcitrant to treatment. We describe a patient with recurrence of KD three times, approximately four years apart. A 10-year-old female with two previous episodes of KD, at 11 months and five years of age), in which she met five out of five criteria for KD and had no coronary involvement, presented with 15 days of fever, conjunctivitis and mucocutaneous changes. Infectious work-up was negative, and she was diagnosed with incomplete KD meeting three out of five criteria. An echocardiogram (ECHO) on day 12 revealed dilation of the right coronary artery (RCA) and left coronary artery (LCA). Treatment with intravenous immunoglobulin (IVIG) and high-dose aspirin was started at an outside hospital. After transfer, serial ECHOs showed evolving coronary aneurysms, left anterior descending (LAD) z-score + 8.2 and RCA z-score + 4.0. She received 10 mg/kg infliximab (day 18) and began clopidogrel. A cardiac MRI (day 20) demonstrated progression of the LAD aneurysm, with a z-score + 13, and warfarin was started. To our knowledge, this is the first report of recurrent KD occurring three times at ~5 year intervals.

Effect of variations in dietary Pi intake on intestinal Pi transporters (NaPi-IIb, PiT-1, and PiT-2) and phosphate-regulating factors (PTH, FGF-23, and MEPE).

Hyperphosphatemia is a common condition in patients with chronic kidney disease (CKD) and can lead to bone disease, vascular calcification, and increased risks of cardiovascular disease and mortality. Inorganic phosphate (Pi) is absorbed in the intestine, an important step in the maintenance of homeostasis. In CKD, it is not clear to what extent Pi absorption is modulated by dietary Pi. Thus, we investigated 5/6 nephrectomized (Nx) Wistar rats to test whether acute variations in dietary Pi concentration over 2 days would alter hormones involved in Pi metabolism, expression of sodium-phosphate cotransporters, apoptosis, and the expression of matrix extracellular phosphoglycoprotein (MEPE) in different segments of the small intestine. The animals were divided into groups receiving different levels of dietary phosphate: low (Nx/LPi), normal (Nx/NPi), and high (Nx/HPi). Serum phosphate, fractional excretion of phosphate, intact serum fibroblast growth factor 23 (FGF-23), and parathyroid hormone (PTH) were significantly higher and ionized calcium was significantly lower in the Nx/HPi group than in the Nx/LPi group. The expression levels of NaPi-IIb and PiT-1/2 were increased in the total jejunum mucosa of the Nx/LPi group compared with the Nx/HPi group. Modification of Pi concentration in the diet affected the apoptosis of enterocytes, particularly with Pi overload. MEPE expression was higher in the Nx/HPi group than in the Nx/NPi. These data reveal the importance of early control of Pi in uremia to prevent an increase in serum PTH and FGF-23. Uremia may be a determining factor that explains the expressional modulation of the cotransporters in the small intestine segments.

Treatment of Pancreatic Cancer Patient-Derived Xenograft Panel with Metabolic Inhibitors Reveals Efficacy of Phenformin.

Purpose: To identify effective metabolic inhibitors to suppress the aggressive growth of pancreatic ductal adenocarcinoma (PDAC), we explored the in vivo antitumor efficacy of metabolic inhibitors, as single agents, in a panel of patient-derived PDAC xenograft models (PDX) and investigated whether genomic alterations of tumors correlate with the sensitivity to metabolic inhibitors.Experimental Design: Mice with established PDAC tumors from 6 to 13 individual PDXs were randomized and treated, once daily for 4 weeks, with either sterile PBS (vehicle) or the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), transaminase inhibitor aminooxyacetate (AOA), pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA), autophagy inhibitor chloroquine (CQ), and mitochondrial complex I inhibitor phenformin/metformin.Results: Among the agents tested, phenformin showed significant tumor growth inhibition (>30% compared with vehicle) in 5 of 12 individual PDXs. Metformin, at a fivefold higher dose, displayed significant tumor growth inhibition in 3 of 12 PDXs similar to BPTES (2/8 PDXs) and DCA (2/6 PDXs). AOA and CQ had the lowest response rates. Gene set enrichment analysis conducted using the baseline gene expression profile of pancreatic tumors identified a gene expression signature that inversely correlated with phenformin sensitivity, which is in agreement with the phenformin gene expression signature of NIH Library of Integrated Network-based Cellular Signatures (LINCS). The PDXs that were more sensitive to phenformin showed a baseline reduction in amino acids and elevation in oxidized glutathione. There was no correlation between phenformin response and genetic alterations in KRAS, TP53, SMAD4, or PTENConclusions: Phenformin treatment showed relatively higher antitumor efficacy against established PDAC tumors, compared with the efficacy of other metabolic inhibitors and metformin. Phenformin treatment significantly diminished PDAC tumor progression and prolonged tumor doubling time. Overall, our results serve as a foundation for further evaluation of phenformin as a therapeutic agent in pancreatic cancer. Clin Cancer Res; 23(18); 5639-47. ©2017 AACR.

Atrial Thrombus in a Neonate: A Diagnostic Challenge.

Introduction Left atrial thrombus is a rare finding in a neonate. In the previous literature, atrial thrombi have been associated with catheter placement or congenital heart disease in a preterm infant. Case We report the case of a full-term neonate with no known risk factors found to have a left atrial thrombus. The neonate was born at 38 weeks' gestation to a 31-year-old female via cesarean section who was sent to the normal nursery. On postnatal day 5, the infant was noted to have low-to-medium level of oxygen saturations (∼90%) and was transferred to the neonatal intensive care unit with an echocardiogram completed on postnatal day 6 demonstrating a mobile, pedunculated mass attached to the left atrial septum with an appearance concerning for atrial myxoma. The infant underwent surgical resection on postnatal day 8 and pathology revealed the mass to be a left atrial thrombus. Discussion The rare finding of an atrial thrombus in a neonate has previously been associated with central venous catheter placement or congenital heart disease. This case is unusual in that the patient had neither condition. Although echocardiogram findings appeared more consistent with atrial myxoma, final pathology revealed a left atrial thrombus. Additionally, hypercoagulability work-up for this neonate was largely negative. This report underscores the importance of identification, search for etiology, and prompt therapy to prevent potential catastrophic outcomes.

Notch signaling pathway targeted therapy suppresses tumor progression and metastatic spread in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) remains a lethal human malignancy with historically limited success in treatment. The role of aberrant Notch signaling, which requires the constitutive activation of γ-secretase, in the initiation and progression of PDA is well defined and inhibitors of this pathway are currently in clinical trials. Here we investigated the in vivo therapeutic effect of PF-03084014, a selective γ-secretase inhibitor, alone and in combination with gemcitabine in pancreatic cancer xenografts. PF-03084014 treatment inhibited the cleavage of nuclear Notch 1 intracellular domain and Notch targets Hes-1 and Hey-1. Gemcitabine treatment showed good response but not capable of inducing tumor regressions and targeting the tumor-resident cancer stem cells (CD24(+)CD44(+) and ALDH(+) tumor cells). A combination of PF-03084014 and gemcitabine treatment resulted tumor regression in 3 of 4 subcutaneously implanted xenograft models. PF-03084014, and in combination with gemcitabine reduced putative cancer stem cells, indicating that PF-03084014 target the especially dangerous and resilient cancer stem cells within pancreatic tumors. Tumor re-growth curves plotted after drug treatments demonstrated that the effect of the combination therapy was sustainable than that of gemcitabine. Notably, in a highly aggressive orthotopic model, PF-03084014 and gemcitabine combination was effective in inducing apoptosis, inhibition of tumor cell proliferation and angiogenesis, resulting in the attenuation of primary tumor growth as well as controlling metastatic dissemination, compared to gemcitabine treatment. In summary, our preclinical data suggest that PF-03084014 has greater anti-tumor activity in combination with gemcitabine in PDA and provides rationale for further investigation of this combination in PDA.

The gamma secretase inhibitor MRK-003 attenuates pancreatic cancer growth in preclinical models.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, with most patients facing an adverse clinical outcome. Aberrant Notch pathway activation has been implicated in the initiation and progression of PDAC, specifically the aggressive phenotype of the disease. We used a panel of human PDAC cell lines as well as patient-derived PDAC xenografts to determine whether pharmacologic targeting of Notch pathway could inhibit PDAC growth and potentiate gemcitabine sensitivity. MRK-003, a potent and selective γ-secretase inhibitor, treatment resulted in the downregulation of nuclear Notch1 intracellular domain, inhibition of anchorage-independent growth, and reduction of tumor-initiating cells capable of extensive self-renewal. Pretreatment of PDAC cells with MRK-003 in cell culture significantly inhibited the subsequent engraftment in immunocompromised mice. MRK-003 monotherapy significantly blocked tumor growth in 5 of 9 (56%) PDAC xenografts. A combination of MRK-003 and gemcitabine showed enhanced antitumor effects compared with gemcitabine in 4 of 9 (44%) PDAC xenografts, reduced tumor cell proliferation, and induced both apoptosis and intratumoral necrosis. Gene expression analysis of untreated tumors indicated that upregulation of NF-κB pathway components was predictive of sensitivity to MRK-003, whereas upregulation in B-cell receptor signaling and nuclear factor erythroid-derived 2-like 2 pathway correlated with response to the combination of MRK-003 with gemcitabine. Our findings strengthen the rationale for small-molecule inhibition of Notch signaling as a therapeutic strategy in PDAC.

Fasciolose hepática

A fasciolose hepática é uma zoonose causada pela Fasciola hepatica, verme achatado e de corpo foliáceo, que tem ampla distribuição geográfica e é conhecido popularmente como baratinha do fígado ou saguaipé. F. hepatica é um trematódeo encontrado no fígado e canais biliares de animais de sangue quente, ocorrendo em ovinos, caprinos, bovinos, búfalos, suínos e em seres humanos

Tumor engraftment in nude mice and enrichment in stroma- related gene pathways predict poor survival and resistance to gemcitabine in patients with pancreatic cancer.

PURPOSE: The goal of this study was to evaluate prospectively the engraftment rate, factors influencing engraftment, and predictability of clinical outcome of low-passage xenografts from patients with resectable pancreatic ductal adenocarcinoma (PDA) and to establish a bank of PDA xenografts. EXPERIMENTAL DESIGN: Patients with resectable PDA scheduled for resection at the Johns Hopkins Hospital were eligible. Representative pieces of tumor were implanted in nude mice. The status of the SMAD4 gene and content of tumor-generating cells were determined by immunohistochemistry. Gene expression was carried out by using a U133 Plus 2.0 array. Patients were followed for progression and survival. RESULTS: A total of 94 patients with PDA were resected, 69 tumors implanted in nude mice, and 42 (61%) engrafted. Engrafted carcinomas were more often SMAD4 mutant, and had a metastatic gene expression signature and worse prognosis. Tumors from patients resistant to gemcitabine were enriched in stroma-related gene pathways. Tumors sensitive to gemcitabine were enriched in cell cycle and pyrimidine gene pathways. The time to progression for patients who received treatment with gemcitabine for metastatic disease (n = 7) was double in patients with xenografts sensitive to gemcitabine. CONCLUSION: A successful xenograft was generated in 61% of patients attempted, generating a pool of 42 PDA xenografts with significant biological information and annotated clinical data. Patients with PDA and SMAD4 inactivation have a better engraftment rate. Engraftment is a poor prognosis factor, and engrafted tumors have a metastatic gene expression signature. Tumors from gemcitabine-resistant patients were enriched in stromal pathways.

A pilot clinical study of treatment guided by personalized tumorgrafts in patients with advanced cancer.

Patients with many advanced solid cancers have very poor prognosis, and improvements in life expectancy are measured only in months. We have recently reported the remarkable clinical outcome of a patient with advanced, gemcitabine-resistant, pancreatic cancer who was later treated with DNA-damaging agents, on the basis of the observation of significant activity of this class of drugs against a personalized tumorgraft generated from the patient's surgically resected tumor. Here, we extend the approach to patients with other advanced cancers. Tumors resected from 14 patients with refractory advanced cancers were propagated in immunodeficient mice and treated with 63 drugs in 232 treatment regimens. An effective treatment regimen in the xenograft model was identified for 12 patients. One patient died before receiving treatment, and the remaining 11 patients received 17 prospectively guided treatments. Fifteen of these treatments resulted in durable partial remissions. In 2 subjects, no effective treatments were found. Overall, there was a remarkable correlation between drug activity in the model and clinical outcome, both in terms of resistance and sensitivity. The data support the use of the personalized tumorgraft model as a powerful investigational platform for therapeutic decision making and to efficiently guide cancer treatment in the clinic.

MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts.

PURPOSE: Investigate the efficacy and pharmacodynamic effects of MK-1775, a potent Wee1 inhibitor, in both monotherapy and in combination with gemcitabine (GEM) using a panel of p53-deficient and p53 wild-type human pancreatic cancer xenografts. EXPERIMENTAL DESIGN: Nine individual patient-derived pancreatic cancer xenografts (6 with p53-deficient and 3 with p53 wild-type status) from the PancXenoBank collection at Johns Hopkins were treated with MK-1775, GEM, or GEM followed 24 hour later by MK-1775, for 4 weeks. Tumor growth rate/regressions were calculated on day 28. Target modulation was assessed by Western blotting and immunohistochemistry. RESULTS: MK-1775 treatment led to the inhibition of Wee1 kinase and reduced inhibitory phosphorylation of its substrate Cdc2. MK-1775, when dosed with GEM, abrogated the checkpoint arrest to promote mitotic entry and facilitated tumor cell death as compared to control and GEM-treated tumors. MK-1775 monotherapy did not induce tumor regressions. However, the combination of GEM with MK-1775 produced robust antitumor activity and remarkably enhanced tumor regression response (4.01-fold) compared to GEM treatment in p53-deficient tumors. Tumor regrowth curves plotted after the drug treatment period suggest that the effect of the combination therapy is longer-lasting than that of GEM. None of the agents produced tumor regressions in p53 wild-type xenografts. CONCLUSIONS: These results indicate that MK-1775 selectively synergizes with GEM to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts.

Antitumor effects and biomarkers of activity of AZD0530, a Src inhibitor, in pancreatic cancer.

PURPOSE: To determine the efficacy of AZD0530, an orally active small molecule Src inhibitor, in human pancreatic cancer xenografts and to seek biomarkers predictive of activity. EXPERIMENTAL DESIGN: Sixteen patient-derived pancreatic cancer xenografts from the PancXenoBank collection at Johns Hopkins were treated with AZD0530 (50 mg/kg/day, p.o.) for 28 days. Baseline gene expression profiles of differently expressed genes in 16 tumors by Affymetrix U133 Plus 2.0 gene array were used to predict AZD0530 sensitivity in an independent group of eight tumors using the K-Top Scoring Pairs (K-TSP) method. RESULTS: Three patient tumors of 16 were found to be sensitive to AZD0530, defined as tumor growth <50% compared with control tumors (100%). Western blot and/or immunohistochemistry results showed that AZD0530 administration resulted in the down-regulation of Src, FAK, p-FAK, p-paxillin, p-STAT-3, and XIAP in sensitive tumor xenografts compared with control tumors. The K-TSP classifier identified one gene pair (LRRC19 and IGFBP2) from the 16 training cases based on a decision rule. The classifier achieved 100% and 83.3% of sensitivity and specificity in an independent test set that consists of eight xenograft cases. CONCLUSIONS: AZD0530 treatment significantly inhibits the tumor growth in a subset of human pancreatic tumor xenografts. One gene pair (LRRC19 and IGFBP2) identified by the K-TSP classifier has high predictive power for AZD0530 sensitivity, suggesting the potential for this gene pair as biomarker for pancreatic tumor sensitivity to AZD0530.

Effect of bioactive peptides isolated from yeastolate, lactalbumin and NZCase in the insect cell growth.

In this study, we have described the biological activity of various hydrolysates and its effect on cell growth, growth rate and doubling time. A potent cell culture enhancer factor was observed in the yeastolate hydrolysates, mainly in the protein fractions with low molecular weight. In this case, a growth enhancer of 60.66% was obtained. Despite a lower efficiency of crude lactalbumin hydrolysates (14%), when lactalbumin and yeastolate were added together to the culture, the cell yields were of 102%, showing a synergic effect. Nevertheless, sub fraction from LMW, of lactalbumin, obtained by Sephadex G-10 gel filtration chromatography showed a higher positive effect (23.3%) than low molecular weight fraction of lactalbumin without this chromatography step (11.3%). It is suggested that low molecular weight lactalbumin could have some inhibitory protein. On the other hand, NZCase low molecular weight showed a positive effect of 29.33%, while its sub fractions showed a negative effect of 5.5%. With these data we can suggest that these hydrolysates could be an important element to design new media, serum free, being helpful in protein recombinant production.

Salivary analysis of patients with chronic renal failure undergoing hemodialysis.

This study evaluated the flow rate and composition of whole saliva in patients with chronic renal failure undergoing hemodialysis. In the group on dialysis (RG) (n = 15), saliva was collected just prior to hemodialysis CT1) and at completion (T2), while in the healthy subjects (HG)(n = 15) saliva was collected at the same time of day as the pre-dialysis. Saliva samples were analyzed by inductively coupled argon plasma with atomic emission spectrometry. Significant differences were found in the flow rate, potassium, magnesium and phosphorus concentrations at the RG-T1 and HG (p < 0.05). Sodium concentration at RG-T1 and RG-T2 were higher than HG (p < 0.05). Total protein concentration was higher at RG-T1 than at the other two analyses. Salivary peroxidase activity at RG-T1 and RG-T2 was lower than at HG. Our findings suggest that in patients with chronic renal failure, the saliva is altered. Hemodialysis, however, seems to help control saliva composition and flow rate.

Co-localization of nestin and insulin and expression of islet cell markers in long-term human pancreatic nestin-positive cell cultures.

Strategies to differentiate progenitor cells into beta cells in vitro have been considered as an alternative to increase beta cell availability prior to transplantation. It has recently been suggested that nestin-positive cells could be multipotential stem cells capable of expressing endocrine markers upon specific stimulation; however, this issue still remains controversial. Here, we characterized short- and long-term islet cell cultures derived from three different human islet preparations, with respect to expression of nestin and islet cell markers, using confocal microscopy and semi-quantitative RT-PCR. The number of nestin-positive cells was found to be strikingly high in long-term cultures. In addition, a large proportion (49.7%) of these nestin-positive cells, present in long-term culture, are shown to be proliferative, as judged by BrdU incorporation. The proportion of insulin-positive cells was found to be high in short-term (up to 28 days) cultures and declined thereafter, when cells were maintained in the presence of 10% serum, concomitantly with the decrease in insulin and PDX-1 expression. Interestingly, insulin and nestin co-expression was observed as a rare event in a small proportion of cells present in freshly isolated human islets as well as in purified islet cells cultured in vitro for long periods of time. In addition, upon long-term subculturing of nestin-positive cells in 10% serum, we observed reappearance of insulin expression at the mRNA level; when these cultures were shifted to 1% serum for a month, expression of insulin, glucagon and somatostatin was also detected, indicating that manipulating the culture conditions can be used to modulate the nestin-positive cell's fate. Attempts to induce cell differentiation by plating nestin-positive cells onto Matrigel revealed that these cells tend to aggregate to form islet-like clusters, but this is not sufficient to increase insulin expression upon short-term culture. Our data corroborate previous findings indicating that, at least in vitro, nestin-positive cells may undergo the early stages of differentiation to an islet cell phenotype and that long-term cultures of nestin-positive human islet cells may be considered as a potential source of precursor cells to generate fully differentiated/ functional beta cells.

Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection.

Prions are composed of an isoform of a normal sialoglycoprotein called PrP(c), whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K(d) of 10(-7) M. The interaction sites were mapped to amino acids 113-128 from PrP(c) and 230-245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP(c) binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP(c) interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.

Growth and Nutrition of Mollicute-Infected Maize.

Maize bushy stunt phytoplasma (MBSP) and corn stunt spiroplasma (CSS) diseases are widespread in Brazil. The leafhopper Dalbulus maidis is the insect vector for these pathogenic mollicutes. The effects of these diseases on the development of maize plants and the possible interaction of soil water availability on these effects were evaluated in two experiments carried out on potted plants. Experiment 1 was carried out in a 2 × 4 factorial, where factor 1 corresponded to healthy and mollicute-infected plants and factor 2 to the maintenance of 40, 60, 80, and 100% of the total soil water availability. Leafhoppers collected from a field with high incidence of these diseases were used to inoculate plants with mollicutes. There were three treatments in experiment 2: healthy plants, plants infected with phytoplasma, and plants infected with spiroplasma. MBSP was predominant in experiment 1. The infected plants grew less and lowered nutrient uptake, in distinct proportions, indicating a differential effect of mollicutes on nutrient uptake independent of available soil water. Soil water availability did not significantly affect plant growth and nutrient uptake or mollicute infection. The results indicated that plants infected by mollicutes contained less protein than healthy plants. Experiment 2 showed a reduction in growth of plants infected with mollicutes and less nutrient uptake by spiroplasma-infected plants. The results showed a detrimental effect of the spiroplasma on Mg uptake. Both experiments showed more water retention by infected plants than by healthy ones. These experiments clearly demonstrated that reduced plant growth and nutrient uptake are major effects on plants infected with MBSP and CSS.