Short-course High-dose Liposomal Amphotericin B for Human Immunodeficiency Virus-associated Cryptococcal Meningitis: A Phase 2 Randomized Controlled Trial.

Background: We performed a phase 2 noninferiority trial examining the early fungicidal activity (EFA) of 3 short-course, high-dose liposomal amphotericin B (L-AmB) regimens for cryptococcal meningitis (CM) in Tanzania and Botswana. Methods: Human immunodeficiency virus (HIV)-infected adults with CM were randomized to (i) L-AmB 10 mg/kg on day 1 (single dose); (ii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on day 3 (2 doses); (iii) L-AmB 10 mg/kg on day 1 and 5 mg/kg on days 3 and 7 (3 doses); or (iv) L-AmB 3 mg/kg/day for 14 days (control). All patients also received oral fluconazole 1200 mg/day for 14 days. Primary endpoint was mean rate of clearance of cerebrospinal fluid cryptococcal infection (EFA). Noninferiority was defined as an upper limit of the 2-sided 95% confidence interval (CI) of difference in EFA between intervention and control <0.2 log10 colony-forming units (CFU)/mL/day. Results: Eighty participants were enrolled. EFA for daily L-AmB was -0.41 log10 CFU/mL/day (standard deviation, 0.11; n = 17). Difference in mean EFA from control was -0.11 (95% CI, -.29 to .07) log10 CFU/mL/day faster with single dose (n = 16); -0.05 (95% CI, -.20 to .10) log10 CFU/mL/day faster with 2 doses (n = 18); and -0.13 (95% CI, -.35 to .09) log10 CFU/mL/day faster with 3 doses (n = 18). EFA in all short-course arms was noninferior to control. Ten-week mortality was 29% (n = 23) with no statistical difference between arms. All arms were well tolerated. Conclusions: Single-dose 10 mg/kg L-AmB was well tolerated and led to noninferior EFA compared to 14 days of 3 mg/kg/day L-AmB in HIV-associated CM. Induction based on a single 10 mg/kg L-AmB dose is being taken forward to a phase 3 clinical endpoint trial. Clinical Trials Registration: ISRCTN 10248064.

MiR-181a promotes growth of thyroid cancer cells by targeting tumor suppressor RB1.

OBJECTIVE: MicroRNAs (miRs) are critical regulators in cancer development and progression. The current study aimed to investigate the expression and potential function of miR-181a in thyroid cancer. PATIENTS AND METHODS: A total of 15 paired thyroid cancer tissues and adjacent normal tissues were subjected to Real-time Polymerase Chain Reaction (PCR) to evaluate miR-181a expression. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, enzyme linked immunosorbent assay (ELISA) or flow cytometry was employed to assess the growth activity, apoptosis and cell cycle, respectively, upon modulation of the miR-181a expression in TPC-1 cells. Western blot was used to assess protein expression. The interaction between miR-181a and RB1 was tested by luciferase activity assay. RESULTS: The expression of miR-181a was significantly upregulated in thyroid cancer tissues compared with the adjacent tissues. Inhibition of miR-181a attenuated cell growth, which could be abrogated by miR-181a co-transfection. MiR-181a overexpression reduced apoptosis and promoted cell cycle progression; inhibition of miR-181a exerted opposite effects on both cell cycle and apoptosis. MiR-181a directly suppressed RB1 expression. RB1 expression in tumor tissues was downregulated and negatively correlated with miR-181a expression. CONCLUSIONS: miR-181a plays an oncogenic role in thyroid cancer; by targeting RB1, it promotes cell cycle progression and inhibits apoptosis.

Inter-generational effects of the in vitro maturation technique on pregnancy outcomes, early development, and cognition of offspring in mouse model.

In vitro maturation (IVM) of oocytes has been a highly successful method for avoiding the occurrence of severe ovarian hyperstimulation syndrome in some patients during in vitro fertilization. However, the safety of the protocol, especially the long-term effects, is still an issue of debate. The current study is to investigate the long-term effects of IVM on mice through two generations and reveal its inter-generational effects as well. The data indicate that the rates of embryo resorption and fetal death in the F1 generation were significantly increased while the newborn survival rate in the F1 and F2 generations were significantly decreased in the IVM group. Increased body weights in the F1 generation and mouse number per litter in the F2 generation were observed in both the IVM and VVM groups; however, no insulin resistance was detected. No significant differences were detected in birth defects, organ weights, testis histology and sperm motility, estrous cycle, and cognition among the IVM, VVM and N mice in either the F1 or F2 generations. Our results suggest that mouse IVM can affect pregnancy outcomes throughout two generations. IVM does not appear to influence the development and cognition of the offspring throughout two generations.

Assessment of treatment patterns and patient outcomes before vs after implementation of a severity-based Clostridium difficile infection treatment policy.

BACKGROUND: National guidelines recommend oral vancomycin for severe Clostridium difficile infection (CDI) based on results from recent clinical trials demonstrating improved clinical outcomes. However, real-world data to support these clinical trials are scant. AIM: To compare treatment patterns and patient outcomes of those treated for CDI before and after implementation of a severity-based CDI treatment policy at a tertiary teaching hospital. METHODS: This study evaluated adult patients with a positive C. difficile toxin before and after implementation of a policy where patients with severe CDI given metronidazole were switched to oral vancomycin unless contra-indicated. Patients were stratified according to disease severity using a modified published severity score. Treatment patterns based on CDI severity and rates of refractory CDI were assessed. FINDINGS: In total, 256 patients with CDI (mean age 66 years, standard deviation 17, 52% female) were evaluated (before implementation: N = 144; after implementation: N = 112). Use of oral vancomycin for severe CDI increased significantly from 14% (N = 8) to 91% (N = 48) following implementation of the policy (P < 0.0001). Refractory disease in patients with severe CDI decreased significantly from 37% to 15% following implementation of the policy (P = 0.035). No significant differences were noted among patients with mild to moderate CDI. CONCLUSION: A severity-based CDI treatment policy at a tertiary teaching hospital increased the use of oral vancomycin and was associated with decreased rates of refractory CDI.

An absorbed dose map of bone tissue treated with a radiopharmaceutical in vivo.

A beagle humerus treated with Ho-chelate radiopharmaceutical in vivo was examined by electron paramagnetic resonance (EPR) dosimetry. The bone was sectioned and the absorbed dose to each bone fragment was determined by additive re-irradiation of the bone tissue with calibrated doses of gamma radiation. The measured doses ranged from 4.3 Gy to 62 Gy. The highest doses were recorded in the predominately trabecular bone tissue and the lowest doses in the predominately cortical bone tissue. The mean absorbed dose for the entire bone was 17 Gy. The data from 50 bone fragments were combined to create an absorbed dose map of the interior bone surface.

Plasmons in the metallic nanoparticle-film system as a tunable impurity problem.

We show that the plasmon resonances of a metallic nanoparticle interacting with the surface plasmons of a metallic film is an electromagnetic analogue of the spinless Anderson-Fano model. This is the same model used to describe the interaction of a localized electronic state with a continuous band of electronic states. The three characteristic regimes of this model are realized here, where the energy of the nanoparticle plasmon resonance lies above, within, or below the energy band of surface plasmon states. These three interaction regimes are controlled by film thickness. The latter regime is experimentally observed and identified.

[A research on the correlation of oral mucosal patches stria diseases and epithelial dysplasia].

OBJECTIVE: The aim of the present study was to elucidate the relationship between oral mucosal patches-stria diseases and epithelial dysplasia. METHODS: Three hundred and six cases were studied, including one hundred and forty cases of leukoplakia, one hundred and twenty cases of lichen planus,forty four cases of discoid lupus erythematosus and two cases of erythroplakia. The incidence of epithelial dysplasia in each kind of mucosal patches stria diseases were analyzed and compared. The developing tendency of 30 cases which underwent biopsy more than two times were analyzed and observed. RESULTS: In the common mucosal patches-stria diseases,the incidence of epithelial dysplasia in leukoplakia was 56.43%,lichen planus was 20.83%, discoid lupus erythematosus was 13.64%, erythroplakia was 100%. The incidence of epithelial dysplasia were detected significantly more often in leukoplakia and erythroplakia than in lichen planus and discoid lupus erythematosus (P<0.05). CONCLUSION: Leukoplakia and erythroplakia belong to premalignant lesion,whether lichen planus belongs to premalignant lesion or not is controversial.

The role of disulfide-linked dimerization in interleukin-3 receptor signaling and biological activity.

Cysteine residues 86 and 91 of the beta subunit of the human interleukin (hIL)-3 receptor (hbetac) participate in disulfide-linked receptor subunit heterodimerization. This linkage is essential for receptor tyrosine phosphorylation, since the Cys-86 --> Ala (Mc4) and Cys-91 --> Ala (Mc5) mutations abolished both events. Here, we used these mutants to examine whether disulfide-linked receptor dimerization affects the biological and biochemical activities of the IL-3 receptor. Murine T cells expressing hIL-3Ralpha and Mc4 or Mc5 did not proliferate in hIL-3, whereas cells expressing wild-type hbetac exhibited rapid proliferation. However, a small subpopulation of cells expressing each mutant could be selected for growth in IL-3, and these proliferated similarly to cells expressing wild-type hbetac, despite failing to undergo IL-3-stimulated hbetac tyrosine phosphorylation. The Mc4 and Mc5 mutations substantially reduced, but did not abrogate, IL-3-mediated anti-apoptotic activity in the unselected populations. Moreover, the mutations abolished IL-3-induced JAK2, STAT, and AKT activation in the unselected cells, whereas activation of these molecules in IL-3-selected cells was normal. In contrast, Mc4 and Mc5 showed a limited effect on activation of Erk1 and -2 in unselected cells. These data suggest that whereas disulfide-mediated cross-linking and hbetac tyrosine phosphorylation are normally important for receptor activation, alternative mechanisms can bypass these requirements.

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation.

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMRalpha) and a common signal-transducing beta-subunit (hbetac) that is shared with the interleukin-3 and -5 receptors. We have previously identified a constitutively active extracellular point mutant of hbetac, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287). This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMRalpha (mGMRalpha) subunit, since introduction of mGMRalpha, but not hGMRalpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence. Experiments utilizing mouse/human chimeric GMRalpha subunits indicated that the species specificity lies in the extracellular domain of GMRalpha. Importantly, the requirement for mGMRalpha correlated with the ability of I374N (but not wild-type hbetac) to constitutively associate with mGMRalpha. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMRalpha surface expression. Taken together, these findings suggest a critical role for association with GMRalpha in the constitutive activity of I374N.

[Expression of p53 protein and PCNA in premalignant lesions of oral mucosa and its clinical significance]

OBJECTIVE:The study was to investigate the expression features of p53 protein and PCNA antigens in premalignant lesions of oral mucosa and further to discuss its clinical significance.METHODS: 50 samples were retrieved from the biopsy of the patients with suspicious premalignancy.Followed by the diagnosis pathologically,the sections were labeled with p53 and PCNA antigens by immunohistochemistry. RESULTS: In premalignant lesions,the progression staining with p53 and PCNA antigens were positively corresponding to the epithelial dysplasia from the basal layer to the keratinous layer. CONCLUSION: The p53 and PCNA antigens expressed synchronously in premalignant lesions of oral mucosa and exhibited direct correlation with the degree of epithelial dysplasia;The positive signals with p53 and PCNA antigens in the premalignant lesions may be the earlier incidence in carcinogenesis, which may initiated at the earlier stage of epithelial dysplasia.This phenomenon is deserving of clinicians' attention for discovering the premalignant lesions earlier;The detection of p53 and PCNA antigens would be helpful to find out the lesion which has the tendency turning to dysplasia.

Muscarinic m4 receptor activation by some atypical antipsychotic drugs.

To clarify the findings that clozapine is both a muscarinic receptor agonist and antagonist, we examined the effects of neuroleptics on forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells expressing human muscarinic m4 receptors (CHO-hm4) and in rat striatum. With CHO-hm4 cells, clozapine induced a concentration-dependent and atropine-sensitive inhibition on cAMP formation, with EC50 = 60 nM and Emax = 74% of carbachol maximum. Other atypical neuroleptics, fluperlapine, tenilapine and olanzapine, were similar but less potent, while risperidone, rilapine, quetiapine (ICI 204,636), sertindole, and ziprasidone had almost no effect. Typical neuroleptics, haloperidol, chlorpromazine, fluphenazine, thiothixene, thioridazine, and molindone, showed either no effect or an atropine-resistant inhibition of cAMP formation. However, in rat striatal tissues, clozapine, up to 10 microM, did not show a significant inhibition of cAMP formation, probably due to a relatively low abundance of muscarinic m4 receptors and the presence of multiple types of muscarinic and other receptors, with which clozapine interacts. Nevertheless, muscarinic m4 receptor agonism, to some extent, may be a relevant mechanism for the therapeutic efficacy and side effects of clozapine and some atypical neuroleptics.

Characterization of the genomic structure, promoter region, and a tetranucleotide repeat polymorphism of the human neurotensin receptor gene.

In the present study, we have cloned the human neurotensin receptor (NTR) gene, determined its structure, demonstrated that its promoter is functional in transfection experiments, and identified the start site of transcription and a tetranucleotide repeat polymorphism that locates at less than 3 kilobase pairs from the gene. The gene contains three introns, all in the coding regions. Several differences in genomic clones and previously characterized cDNA sequences are reconciled. The 5' regulatory region, which is rich in presumptive transcription factors, can drive luciferase expression in transfected CHO-K1 cells. Stepwise 5' deletions identify a positive modulator between -782 and -1309 and a negative modulator between -1309 and -1563. Southern blot analyses demonstrate a single copy gene for the NTR. The tetranucleotide repeat polymorphism is highly informative with at least 23 alleles and might serve as a very useful marker for genetic study of the relationship between the NTR and neuropsychiatric disorders.

Changes in atrioventricular conduction properties with refractory-period modulation.

Dofetilide, clofilium, and risotilide, three drugs known to prolong cardiac action potentials and refractory periods, were studied by using a perfused isolated rabbit heart preparation with intermittent premature pacing and bipolar surface electrograms. The rate-related effects of these drugs on atrioventricular (AV) conduction were tested by pacing at a long (400 ms) and a short (250 ms) basic cycle length (BCL). All three drugs increased refractory periods in a concentration-dependent manner in most segments of the AV axis. The maximal atrio-His (AH) conduction interval (AHmax) and delta AH (AHmax - AHmin) produced by premature pacing was decreased by the highest concentration of each drug at the 400-ms BCL, whereas only clofilium reduced AHmax and delta AH at the 250-ms BCL. Changes in delta AH correlated best with changes in the atrial functional refractory period. The His-Purkinje system conduction interval (HV), represented by delta HV, was unaffected by any drug at either BCL. These results show that if atrial or nodal refractory periods are increased sufficiently, AHmax but not AHmin was decreased at the 400-ms BCL. Because dofetilide and risotilide did not affect AHmax at the 250-ms BCL, these drugs may be less effective at preventing AV nodal reentrant tachycardias than a drug such as clofilium that displays less rate dependency.

Characterization and chromosomal localization of the human A2a adenosine receptor gene: ADORA2A.

The gene for the stimulatory G protein-coupled human A2a adenosine receptor was isolated and sequence analysis revealed two exons that are interrupted by an intron of approximately 6.4 kb. An intron is located in the same region in the human A1 and A2b adenosine receptor genes. Comparison of the A2a genomic and cDNA sequences reveals two nucleotide differences in the coding region and the presence of an aberrant sequence in the 5'208 base pairs of the A2a cDNA including a polymorphism in the third base of codon Tyr-361 and Gly codon which was always detected at residue 392, indicated that the Arg codon present in the cDNA may be an artifact. Fluorescent in situ hybridization and PCR analysis of human-hamster hybrid cell panels shows that the A2a receptor gene is localized to chromosome 22q11.2. This is in contrast with previous reports (subsequently retracted) which mapped the A2a gene to chromosome 11q11-13.

Developmental effects of d-sotalol on anterograde and retrograde atrioventricular conduction in the rabbit.

INTRODUCTION: These experiments investigate the developmental effects of d-sotalol on standard electrophysiologic parameters of anterograde and retrograde AV conduction in the rabbit. METHODS AND RESULTS: Using bipolar electrograms and standard pacing techniques, the effects of graded concentrations of d-sotalol on anterograde and retrograde conduction in mature and immature perfused rabbit hearts were compared. Also, a quantitative assessment of the drug's effects on a rate-dependent property of anterograde AV node (AVN) conduction, termed the "recovery process," was compared in mature and immature rabbit hearts. The main developmental electrophysiologic findings of this investigation are: (1) in both the mature and immature rabbit heart, d-sotalol increases the anterograde conduction time and prolongs refractoriness of the AVN, yet the minimal concentrations of d-sotalol that produce these changes are lower in the neonate; (2) d-sotalol increases the anterograde refractory period of the His-Purkinje system in both age groups, but increases anterograde infra-Hisian conduction only in the neonate; (3) 1 x 10-4 M d-sotalol significantly changes the time constant of the AVN recovery process in the neonate, but not in the adult; (4) for retrograde conduction, slow conduction through the AVN (HAmax) and infra-Hisian region (VHmax) are increased by d-sotalol in the neonate, but not in the adult. CONCLUSIONS: The findings of this study illustrate that d-sotalol has different effects on parameters of the developing AV conduction system. This implies that there may be maturational changes in the ionic currents that are responsible for anterograde and retrograde AVN and His-Purkinje conduction.

Exclusion of close linkage of bipolar disorder to the Gs-alpha subunit gene in nine Australian pedigrees.

Growing evidence suggests that guanine nucleotide binding proteins (G proteins) may be involved in both the pathogenesis and treatment of bipolar affective disorder. Both overactive G proteins and increased levels of the alpha subunit of the stimulatory form (Gs-alpha) have been demonstrated in peripheral leucocytes of manic patients while an increase of Gs-alpha subunit levels has also been found in a postmortem study of bipolar disorder. The function of Gs and Gi alpha subunits has now been shown to be affected by lithium. The present study aimed to determine whether bipolar affective disorder was linked to the Gs-alpha subunit gene which has been mapped to chromosomal region 20q13.2. Linkage analysis utilized the PCR amplification of a portion of the Gs-alpha gene that contains a dinucleotide repeat (CA repeat) polymorphism. Linkage of bipolar disorder and recurrent depression to the Gs-alpha subunit gene was tested using a series of autosomal dominant and recessive models with varying penetrance levels. Additionally, linkage was examined using a series of levels of definitions of affective illness. Close linkage to the Gs-alpha subunit gene was strongly excluded using each model and definition. Thus, our study indicates that a genetic defect in the Gs-alpha subunit gene is unlikely to be the cause of bipolar disorder.