Urinary JCV-DNA testing during natalizumab treatment may increase accuracy of PML risk stratification.

The risk of progressive multifocal leukoencephalopathy (PML) in patients treated with natalizumab for multiple sclerosis (MS) is a serious concern. The presence of anti-JC virus antibodies is a risk factor for PML development, but 2.5 % of the patients result falsely-negative, while the prognostic relevance of testing JCV-DNA in biological fluids of treated patients is debated. Aim of this work was to evaluate the utility of testing JCV-DNA, together with anti-JCV antibodies, in biological samples of treated patients as a tool for PML risk stratification. 126 subjects from 5 MS Centers in Italy were included in the study. We performed a cross-sectional study in 63 patients testing JCV-DNA in blood, peripheral blood cells and urine. We longitudinally assessed the presence of JCV-DNA in a cohort of 33 subjects, one of which developed PML. We could test retrospectively serum samples from another PML case occurred during natalizumab therapy. Anti-JCV antibodies and urinary JCV-DNA were both tested in 73 patients. No changes in JCV-DNA status occurred during natalizumab treatment. The subject who developed PML in the longitudinal cohort had detectable JCV-DNA in urine at all time-points while serum or blood from both PML patients were always negative before the onset of disease and, in one case, after. Four subjects with JCV-DNA in urine and undetectable anti-JCV antibodies were retested for anti-JCV antibodies and three out of four resulted positive. In conclusion, testing JCV-DNA in urine is complementary to testing anti-JCV antibodies in identifying patients at risk of PML.

Stability study of lipoic acid in the presence of vitamins A and E in o/w emulsions for cosmetic application.

The effectiveness of any cosmetic product containing a functional ingredient is determined by the skin delivery of the active molecule, which is influenced by the type of carrier and the molecule itself. Furthermore, the functional ingredient should be stable in the formulation. The purpose of this paper is to study the stability of lipoic acid in the presence of vitamins A (as palmitate) and E (as acetate) in semisolids for cosmetic use. The systems formulated were studied in regard to their aspect, pH, stability under centrifugation, and rheological behavior. The chemical analyses of lipoic acid and vitamins A and E were carried out by HPLC after studying the specificity of the method employed in each case. The quantitation of the active principles was performed by HPLC with C18 (5 microm) columns. The mobile phase was methanol for the vitamins, with spectrophotometric detection at 325 nm for vitamin A and 230 nm for vitamin E. The mobile phase for lipoic acid was methanol:water (80:20) and phosphoric acid at pH 3.0, with spectrophotometric detection at 332 nm. All systems were stable to centrifugation, and no significant modification of rheological behavior was observed in relation to the base emulsion used as control. The chemical studies performed indicated that although lipoic acid is not very stable in these formulations, the presence of vitamin A favors its chemical stability.

HPLC analysis of 5H-benzo[a]carbazole with antifungal activity.

A sensitive and simple high-performance liquid chromatographic (HPLC) method for the assay of 6,11-dihydro-2-methoxy-5H-benzo[a]carbazole (1) and 6,11-dihydro-2-methoxy-11-[2-(1-piperidinyl)]ethyl-5H-benzo[a]carbazole (2) was developed. The procedure is based on the use of the reversed-phase high-performance liquid chromatographic (RP-HPLC) method with UV detector. Each analysis required no longer than 11 min. A linear relationship between the concentration of both the drugs and the UV absorbance at 254 nm was obtained. This linearity was maintained over the concentration ranged from 5 to 80 microg/ml. The detection limits were found to be 1.6 and 0.7 ng for compounds 1 and 2. The quantitation limits were found to be 5.3 and 2.5 ng for compounds 1 and 2, respectively. For recovery studies, several determinations were carried out. Recovery values ranged from 98 to 102.1% for compound 1 and from 98.4 to 101.6% for compound 2. Method precision was also evaluated and RSD% found was less than 2%. This method was applied without any interference from degradation products.

Nuclear cathepsin B-like protease cleaves transcription factor YY1 in differentiated cells.

Differentiation of pluripotent cells into differentiated cell types involves changes in many aspects of cellular biochemistry. Many of these changes result in alterations of gene expression, which may occur by changing the activity of transcription factors. The cell line NTERA-2 (NT2) can be differentiated into various cell types by incubation with retinoic acid. The differentiated cell type is also permissive for infection with the human herpesvirus cytomegalovirus (CMV). The transcription factor YY1 has been shown to regulate the immediate-early promoter of CMV in a differentiation specific manner by binding to one site at -958 to -950 and to at least two sites in the enhancer. It is demonstrated here that there is a second YY1 site in the modulator between -995 and -987. Levels of YY1 DNA binding activity and protein decrease in NT2 cells as they are differentiated with retinoic acid. This decrease in protein is due to the degradation of YY1 by a cathepsin B-like activity found in nuclear extracts. The cleavage products of YY1 include the intact C-terminal half of the protein, which contains the zinc fingers and the DNA binding activity. This suggests a mechanism that allows expression of the CMV immediate-early promoter in differentiated cells.

Dissolution test for silymarin tablets and capsules.

Silybine (SBN), isosilybine (ISBN), silycristine (SCN), silydianine (SDN), and taxifoline (TXF) are the main active flavonoids commonly found in the dried fruits of Silybum marianum, Gaertner (Compositae). Concentrations of these compounds, except TXF, are usually expressed together as silymarin content. This paper describes a simple dissolution test developed to estimate silymarin (Sl) in pharmaceutical formulations. Five commercial products were tested using this new method (including tablets, sugar tablets, and capsules): two from Argentina, one from Brazil, one from Spain, and one from Italy. Results demonstrated that, provided the dosage form disintegrates, amounts dissolved range from 50 to 90% of the labeled value. Products were analyzed by high performance liquid chromatography (HPLC) and UV spectrophotometry.

Antitumoral activity of trisubstituted dihydrobenzo(a)carbazoles. Part III.

Two recently synthesized, trisubstituted dihydrobenzo(a)carbazoles were investigated regarding their anti-HIV and antitumoral activity. The compounds showed some activity against melanoma, renal cancer and breast cancer cell lines.

A stability-indicating HPLC method to determine cyproterone acetate in tablet formulations.

A simple and accurate liquid chromatographic method was developed to estimate cyproterone acetate (CA) in pharmaceuticals. The drug was chromatographed on a reversed-phase C18 column. Eluents were monitored at a wavelength of 254 nm utilizing a mixture (60:40) of acetonitrile and water. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method was statistically validated for linearity, accuracy, precision, and selectivity. Due to its simplicity and accuracy, we believe that the method can be used for routine quality control analysis. No specific sample preparation is required except for the use of a column guard and a suitable prefilter attached to the syringe.

A new method for quality control of zinc pyritione pharmaceutical and cosmetic forms.

A simple high-performance liquid chromatographic (HPLC) method was developed to determine zinc pyritione in pharmaceutical and cosmetic products. Reversed-phase chromatography was conducted using a C18 column with an isocratic mobile phase consisting of a suitable mixture of methanol, acetonitrile, and water (30:2.5:20). The effluent was monitored on a ultraviolet (UV) detector at 243 nm. The method was validated following International Conference on Harmonisation (ICH) suggestions and proved accurate, precise, and specific.

Development and validation of a reversed-phase liquid chromatographic method for analysis of estradiol valerate and medroxyprogesterone acetate in a tablet formulation.

A simple and accurate liquid chromatographic method was developed for estimation of estradiol valerate and medroxyprogesterone acetate in pharmaceuticals. Drugs were chromatographed on a reverse phase C18 column, using a mixture (30:70) of ammonium nitrate buffer and acetonitrile and eluants monitored at a wavelength of 280 nm. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method was statistically validated for its linearity, accuracy, precision and selectivity. Due to its simplicity and accuracy, the authors believe that the method may be used for routine quality control analysis. It does not require any specific sample preparation except the use of a column guard before the analytical column and suitable prefilter attached to the syringe prior to injection.

Bioavailability study of paracetamol tablets in saliva and urine.

A bioavailability study of two lots of paracetamol tablets was carried out in 5 healthy volunteers, using a crossover aleatory design, and drug monitoring in urine and saliva by high performance liquid chromatography (HPLC). Results were correlated with those obtained in an in vitro dissolution study. Statistical evaluation of bioavailability parameters indicates that the two formulations may be considered bioequivalent, in spite of differences found during early stages of the absorption process, which were preventable according to an in vitro dissolution study.

Antimicrobial activity of 5,6-dihydrobenzo-[a]-carbazoles. Part II.

A new series of twenty-two 5,6-dihydrobenzo[a]carbazoles was synthesized, some showing good antibacterial activity. The presence and position of substituents seems to be critical for such activity.

Effect of manufacturing technology on the dissolution of ampicillin tablets.

The effect of the manufacturing technology on the dissolution of ampicillin tablets have been analyzed. Several kinetic parameters were then calculated. The dissolution rate of ampicillin increases in tablets prepared by direct compression or dry granulation, with microcrystalline cellulose and colloidal silicon dioxide. Studies of in vitro bioequivalence of the pharmaceutical products available in the Argentine market were performed. We concluded that not all commercial products are equivalent.

Antimicrobial activity of 5,6-dihydrobenzo-[a]-carbazoles.

A new series of 5,6-dihydrobenzo[a]carbazoles was synthesized, some showing good antibacterial activity. The presence of a dialkylamino ethyl chain on the 2-, 3- or 4-O-substituent seems to be critical for such activity.

Identification of a large bent DNA domain and binding sites for serum response factor adjacent to the NFI repeat cluster and enhancer region in the major IE94 promoter from simian cytomegalovirus.

The major immediate-early (MIE) transactivator proteins of cytomegaloviruses (CMV) play a pivotal role in the initiation of virus-host cell interactions. Therefore, cis- and trans-acting factors influencing the expression of these proteins through their upstream promoter-enhancer regions are important determinants of the outcome of virus infection. S1 nuclease analysis and in vitro transcription assays with the MIE (or IE94) transcription unit of simian CMV (SCMV) (Colburn) revealed a single prominent mRNA start site associated with a canonical TATATAA motif. This initiator region lies adjacent to a 2,400-bp 5'-upstream noncoding sequence that encompasses a newly identified 1,000-bp (A+T)-rich segment containing intrinsically bent DNA (domain C), together with the previously described proximal cyclic AMP response element locus (domain A) and a tandemly repeated nuclear factor I binding site cluster (domain B). Deleted MIE reporter gene constructions containing domain A sequences only yield up to 4-fold stronger basal expression in Vero cells than the intact simian virus 40 promoter-enhancer region, and sequences from position -405 to -69 (ENH-A1) added to a minimal heterologous promoter produced a 50-fold increase of basal expression in an enhancer assay. In contrast, neither the nuclear factor I cluster nor the bent DNA region possessed basal enhancer properties and neither significantly modulated the basal activity of the ENH-A1 segment. A second segment of domain A from position -580 to -450 was also found to possess basal enhancer activity in various cell types. This ENH-A2 region contains three copies of a repeated element that includes the 10-bp palindromic sequence CCATATATGG, which resembles the core motif of serum response elements and proved to bind specifically to the cellular nuclear protein serum response transcription factor. Reporter gene constructions containing four tandem copies of these elements displayed up to 13-fold increased basal enhancer activity and 18-fold tetradecanoyl phorbol acetate responsiveness in U937 cells, but an ENH-A2 DNA segment encompassing two of the core serum response transcription factor binding sites failed to respond to serum induction in NIH 3T3 cells. Although there are overall similarities in the organizations of both the MIE enhancers and MIE transcription units among human CMV, SCMV, and murine CMV, the specific arrangements of repetitive motifs are quite different, and the bent DNA and ENH-A2 domains appear to be unique to SCMV.

Localization of the human cytomegalovirus 2.7-kb major early beta-gene transcripts by RNA in situ hybridization in permissive and nonpermissive infections.

During the early phase of a human cytomegalovirus (HCMV) infection, the 2.7-kb early gene is by far the most abundantly transcribed RNA. Using strand-specific 32P or digoxigenin-labeled riboprobes derived from a subgenomic fragment of the HCMV Towne 2.7-kb early gene, we have performed Northern blot analysis and RNA in situ hybridization on human and mouse fibroblasts infected with HCMV and on 23 formalin-fixed paraffin-embedded sections of tissue obtained at autopsy. By Northern blot analysis, expression of the 2.7 kb early gene was found only in permissive infections. In contrast, specific hybridization was detected in both permissive and nonpermissive cells by RNA in situ hybridization. In nonpermissive cells, hybridization was weak and predominantly nuclear. In permissive cells, strong nuclear and cytoplasmic hybridization was noted. Specific hybridization to cells with and without cytopathic changes was detected with the anti-sense probe in CMV infected tissue obtained at autopsy. When the sense riboprobe was employed, no specific hybridization was found under nondenaturing conditions. These results suggest that in situ hybridization with a probe directed at the 2.7-kb early gene is an effective method of detecting both permissive and nonpermissive HCMV infections in different stages of infection and in localizing the expression of the major early gene in various cell types.

In situ detection of human cytomegalovirus immediate-early gene transcripts within cardiac myocytes of patients with HIV-associated cardiomyopathy.

OBJECTIVE: Recent clinical and echocardiographic studies have identified dilated cardiomyopathy in 10-20% of HIV-infected adults. The purpose of this study was to determine the role of cardiotropic cytomegalovirus (CMV) infection in the development of HIV-associated cardiomyopathy. DESIGN: We generated sense and antisense digoxigenin-labeled riboprobes derived from the CMV immediate-early (IE) and delayed-early (DE) genes and applied them retrospectively to endomyocardial biopsy samples and control autopsy cardiac samples from HIV-infected patients. SETTING: Tertiary care, referral hospital. PATIENTS: Twelve consecutive HIV-infected patients with global left ventricular hypokinesis demonstrated on two-dimensional echocardiography; eight randomly selected control autopsy cardiac samples from HIV-infected patients without cardiac disease during life. MEASUREMENTS AND MAIN RESULTS: Of the 12 endomyocardial biopsy specimens, six (50%) were found to have specific myocyte nuclear and perinuclear hybridization for transcripts of the CMV IE gene, consistent with non-permissive or latent infection. Similar patterns were not found in any of the eight autopsy control samples. All six patients presented with unexplained congestive heart failure and had CD4 counts less than 100 x 10(6)/l; all six biopsy samples had immunohistochemical evidence of increased myocardial major histocompatibility complex (MHC) class I expression, a finding typical of non-HIV myocarditis. None of the endomyocardial biopsy samples had characteristic CMV inclusions and no specific hybridization was noted with the DE gene riboprobe, suggesting that no active viral DNA replication was present. Only two of the six patients with myocyte hybridization with the IE riboprobe had clinical evidence of solid organ infection with CMV at the time of cardiovascular presentation. CONCLUSIONS: This study is the first to demonstrate the expression of the IE gene of CMV within myocytes from HIV-infected patients with cardiomyopathy, suggesting a non-permissive infection of myocytes without classical intranuclear inclusions. Myocyte infection may be necessary to trigger cellular and humoral-mediated cardiac injury and may be best identified using in situ hybridization techniques.

Demonstration of cytomegalovirus nucleic acids in the coronary arteries of transplanted hearts.

Cytomegalovirus (CMV) infection has been associated with the development of accelerated arteriosclerosis in heart transplant recipients. This association prompted the authors to examine, using in situ hybridization techniques, the coronary arteries of 19 patients who had received heart transplants for the presence of CMV nucleic acids. Two probes were employed on formalin-fixed paraffin-embedded tissues. The first was an S35-labeled nick translated probe derived from CMV DNA. CMV nucleic acids were identified in the arterial intima of 5 (45%) of 11 transplanted hearts examined with this probe. Hybridization to cells morphologically consistent with endothelial cells, lymphocytes, and smooth muscle cells was identified. Hybridization was not detected in any of the hearts when a control probe, a vector without CMV DNA, was employed. The second probe that was used was a tritium-labeled strand-specific ribo-probe derived from the immediate early (IE1) gene of CMV. Using this second probe, hybridization was detected in the coronary arteries of three of five hearts that were positive with the DNA probe, in one of the hearts that was negative with the DNA probe, and in three (38%) of eight additional transplanted hearts. The pattern of hybridization was the same as that seen with the DNA probe, and no hybridization was detected when a control sense probe for the IE1 gene of CMV was employed. These results suggest that CMV nucleic acids are present in the coronary arteries of heart transplant recipients.

The functionally active IE2 immediate-early regulatory protein of human cytomegalovirus is an 80-kilodalton polypeptide that contains two distinct activator domains and a duplicated nuclear localization signal.

The IE2 region of the human cytomegalovirus (CMV) strain Towne major immediate-early (MIE) gene encodes a transcriptional transactivator that stimulates expression from a variety of heterologous target promoters but specifically down-regulates its own promoter. By immunofluorescence and Western immunoblot analysis with monospecific peptide antisera, we found that human CMV MIE exon 5 encodes four overlapping polypeptides, two present at immediate-early times (80 and 55 kDa) and two others detected only at late times after infection (55 and 40 kDa). However, only the 80-kDa version (579 amino acids), which is derived from the small upstream exons 2 and 3 fused to the intact exon 5 region, was functionally active in both transactivation and autoregulation as assessed by cotransfection experiments. These results confirm the corrected assignment of the coding capacity of the exon 5 region based on amino acid homology with the equivalent IE2 protein from simian CMV (Colburn). In transient DNA transfection assays, IE2 expression plasmids also produced a predominant full-length 80-kDa protein, which was localized in a distinctive reticular pattern in the nucleus. Two short basic nuclear localization signals in IE2 were identified by deletion analysis and by conversion of a test cytoplasmic herpes simplex virus protein into a form that localized in the nucleus after insertion of either of these two human CMV motifs. Functional assays with MIE region plasmids containing deletions or truncations in exon 5 revealed that both transactivation and autoregulation required several distinct domains within the COOH half of the IE2 protein, whereas a region between codons 99 and 194 could be discarded. Three segments at the NH2 end of the protein between codons 1 to 85, 86 to 98, and 195 to 290 were also essential for transactivation but played no role in autoregulation. Finally, in domain swap experiments, GAL4-fusion proteins containing either an NH2-terminal 51-amino-acid domain from exon 3 (codons 25 to 85) or the COOH-terminal 33-amino-acid domain from exon 5 (codons 544 to 579) identified two distinct activator domains from IE2, both of which have acidic characteristics.