The human immunodeficiency virus Vif protein overcomes the inhibitory activity of the APOBEC3G cytidine deaminase by prohibiting its packaging into virions. Inhibition of APOBEC3G encapsidation is paralleled by a reduction of its intracellular level presumably caused by the Vif-induced proteasome-dependent degradation of APOBEC3G.In this report we employed confocal microscopy to study the effects of Vif on the expression of APOBEC3G on a single cell level. HeLa cells dually transfected with Vif and APOBEC3G expression vectors revealed efficient co-expression of the two proteins. Under optimal staining conditions approximately 80% of the transfected cells scored double-positive for Vif and APOBEC3G. However, the proportion of double-positive cells observed in identical cultures varied dependent on the fixation protocol and on the choice of antibodies used ranging from as low as 40% to as high as 80% of transfected cells. Importantly, single-positive cells expressing either Vif or APOBEC3G were observed both with wild type Vif and a biologically inactive Vif variant. Thus, the lack of APOBEC3G in some Vif-expressing cells cannot be attributed to Vif-induced degradation of APOBEC3G. These findings are consistent with our results from immunoblot analyses that revealed only moderate effects of Vif on the APOBEC3G steady state levels. Of note, viruses produced under such conditions were fully infectious demonstrating that the Vif protein used in our analyses was both functional and expressed at saturating levels.Our results suggest that Vif and APOBEC3G can be efficiently co-expressed. Thus, depletion of APOBEC3G from Vif expressing cells as suggested previously is not a universal property of Vif and thus is not imperative for the production of infectious virions. Replication of human immunodeficiency virus type 1 (HIV-1) in most primary cells and some immortalized T cell lines is dependent on the expression of a functional Vif protein. In the absence of Vif, virus replication is restricted by a host factor that was recently identified as CEM15 (now referred to as APOBEC3G)  but whosVif is a 23-kDa basic protein that is expressed late during infection in a Rev-dependent manner . ImmunocLike Vif, APOBEC3G is a cytoplasmic protein. In fact, co-immunoprecipitation analyses demonstrated an interaction of Vif and APOBEC3G in transiently transfected cells ,27,29-32The current study aims at characterizing in more detail the effects of Vif on the expression of human APOBEC3G on a single cell level. The study was initiated because of the apparent discrepancy between the drastic effects of Vif on APOBEC3G reported by Marin et al and our own finding of only moderate effects of Vif on APOBEC3G expression in transiently transfected cells. In our study, Vif was expressed from a subviral construct in a Tat- and Rev-dependent manner while APOBEC3G was expressed either in a Tat-dependent manner from an HIV-1-LTR-based vector or independently from a CMV-promoter-based expression vector. The Tat-dependent APOBEC3G expression vector was used to restrict APOBEC3G expression to cells also expressing Tat (and thus Vif).Confocal microscopic analysis of HeLa cells transiently transfected with Vif and APOBEC3G expression vectors revealed significant variations in the number of double-positive cells in identical samples ranging from as low as 40% to as high as 80% of transfected cells depending on fixation method and antibodies employed. Importantly, the appearance of cells expressing only Vif or APOBEC3G was observed both with wild type Vif and a biologically inactive variant and thus cannot be explained by Vif-induced degradation of APOBEC3G. Finally, despite the efficient co-expression of Vif and APOBEC3G, viruses produced in these cultures were fully infectious. We therefore conclude that the Vif-induced exclusion of APOBEC3G from virus-producing cells reported by Marin et al  does notA number of previous studies reported the efficient Vif-dependent degradation of APOBEC3G by cellular proteasomes ,8,28. HoAn earlier study investigating the coexpression of Vif and APOBEC3G by confocal microscopy concluded that APOBEC3G was virtually excluded from Vif-expressing COS7 cells . To veriIn the experiment shown in figure In addition of measuring the context-dependent expression of APOBEC3G, we also wanted to determine the influence of the fixation procedure on the efficiency of Vif:APOBEC3G co-staining. It is well known that the choice of fixative can affect the ability of a given antibody to recognize a specific epitope on its target protein. Frequently, epitopes are masked because of the folding properties of a protein in vivo or because of pre-existing protein-protein interactions that may compete for antibody binding. To test this possibility we compared a formaldehyde fixation procedure employed previously  with theHeLa cells were transfected with pNL-A1 and pHIV-APO3G at a 1:1 molar ratio. Cells were fixed 24 hr later either with methanol (MeOH) as in figure For a more quantitative analysis and to determine possible effects that arise from the use of different antibodies, we extended the experiment shown in figure To quantify the results, multiple optical fields were analyzed n = 5–10) with a total of at least 100 transfected cells for each parameter. As can be seen in figure –10 with Under optimal conditions, wild type Vif and APOBEC3G were coexpressed in about 80% of transfected cells . Experiments are ongoing to study the differential effects of Vif expressed from pNL-A1 and Vif expressed from a codon-optimized vector on APOBEC3G stability. However, these results could suggest that the effect of Vif on APOBEC3G steady-state levels may be influenced by the context in which Vif is expressed. At any rate, despite our inability to observe Vif-dependent cellular depletion of APOBEC3G, we were invariably able to recover fully infectious HIV under conditions were the intracellular levels of APOBEC3G were only moderately affected. We therefore conclude that (i) Vif has the ability to rescue viral infectivity even in the presence of APOBEC3G and (ii) that intracellular depletion of APOBEC3G and rescue of viral infectivity may be functionally separable activities of Vif.For now, the reason for the differences in the sensitivity of APOBEC3G to Vif noted by us versus other research groups remains unexplained. APOBEC3G can form oligomeric structures and is able to interact with Vif. It is therefore possible that such complexes undergo conformational changes that can mask epitopes thus limiting the access of antibodies used in the experiments. Thus, the discrepancy between our findings of the coexpression of Vif and APOBEC3G in the majority of cells and the virtual exclusion of APOBEC3G from Vif-expressing cells reported by Marin et al.  may be aThe inability of Vif expressed from pNL-A1 to deplete APOBEC3G is consistent with our previous inability to observe APOBEC3G degradation in kinetic studies . More reExpression of Vif and APOBEC3G in our experimental setup does not lead to the elimination of APOBEC3G from Vif expressing cells. In fact, more than 80% of successfully transfected cells efficiently co-expressed both proteins. Similar results were observed when a biologically inactive Vif variant was co-expressed with APOBEC3G suggesting that the absence of APOBEC3G in some of the Vif-positive cells is not due to Vif-mediated APOBEC3G degradation but reflects a general characteristic of the transient expression system. Moreover, APOBEC3G expression levels were very similar for Vif-positive and Vif-negative cells as judged from the immunostaining consistent with the only modest reduction in APOBEC3G steady-state levels observed in our immunoblot analyses. Nevertheless, viruses produced under such conditions were fully infectious in the presence but not in the absence of Vif attesting to the biological activity of all the proteins involved and demonstrating that Vif was expressed at saturating levels. We conclude that production of infectious viruses from APOBEC3G expressing cells is dependent on Vif but does not necessitate APOBEC3G exclusion from virus-producing cells.gag and pol products. A vif-defective variant of pNL-A1, pNL-A1vif(-) was constructed by deletion of an NdeI/PflMI fragment [The full-length molecular clone pNL4-3  was usedSerum from an HIV-positive patient (APS) was used to detect HIV-1-specific capsid (CA) proteins. A monoclonal antibody to Vif (MAb #319) was used for all immunoblot analyses and some of the immunohistochemical analyses as indicated in the text and was obtained from Michael Malim through the NIH AIDS Research and Reference Reagent Program ,35,37 FoHeLa cells were propagated in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS). LuSIV cells are derived from CEMx174 cells and contain a luciferase indicator gene under the control of the SIVmac239 LTR . These c2 flasks to about 80% confluency. Cells were transfected using LipofectAMINE PLUS™  following the manufacturer's recommendations. A total of 5–6 μg of plasmid DNA per 25 cm2 flask was used. Cells were harvested 24 hr post-transfection. Transfection efficiency in our analyses was generally 30% to 40%.For transfection of HeLa cells, cells were grown in 25 cmVirus stocks were prepared by transfecting HeLa cells with appropriate plasmid DNAs. Virus-containing supernatants were harvested 24 hr after transfection. Cellular debris was removed by centrifugation  and clarified supernatants were filtered (0.45 μm) to remove residual cellular contaminants. For determination of viral infectivity, unconcentrated filtered viral supernatants were used for the infection of indicator cells. For immunoblot analysis of viral proteins, virus particles (7 ml) were concentrated by ultracentrifugation through 4 ml of 20% sucrose in PBS as described before [7 cells), and mixed with an equal volume of sample buffer . Proteins were solubilized by boiling for 10 to 15 min at 95°C with occasional vortexing of the samples to shear chromosomal DNA. Residual insoluble material was removed by centrifugation . Viral proteins were obtained by boiling concentrated viral pellets in a 1:1 mixture of PBS and sample buffer. Cell lysates and viral extracts were subjected to SDS-PAGE; proteins were transferred to PVDF membranes and reacted with appropriate antibodies as described in the text. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies  and visualized by enhanced chemiluminescence .For immunoblot analysis of intracellular proteins, whole cell lysates were prepared as follows: Cells were washed once with PBS, suspended in PBS (400 μl/105) in a 24-well plate total volume 1.2 to 1.4 ml. Cells were incubated for 24 hours at 37°C. Cells were then harvested and lysed in 150 μl of Promega 1x reporter lysis buffer . To determine the luciferase activity in the lysates, 50 μl of each lysate were combined with luciferase substrate  by automatic injection and light emission was measured for 10 seconds at room temperature in a luminometer .To determine viral infectivity, virus stocks were normalized for equal reverse transcriptase activity and used to infect LuSIV cells  for 10 min followed by two washes in PBS or fixed in FA buffer  for 20 min at room temperature followed by two washes in PBS. Coverslips were stored in PBS at 4°C until use. FA-fixed samples were permeabilized for 30 min at room temperature in permeabilization buffer  prior to incubation with antibodies. For antibody staining, cover slips were incubated in a humid chamber at 37°C for 30 min with primary antibodies at appropriate dilutions in 1% BSA in PBS. Cover slips were washed once in PBS  and incubated with Texas-Red- or Cy2-conjugated secondary antibodies (diluted in 1% BSA in PBS) for 30 min at 37°C in a humid chamber. Cover slips were then washed twice with PBS and mounted onto microscope slides with glycerol gelatin  containing 0.1M N-propyl gallate  to prevent photo bleaching and were stored at 4°C in the dark until analyzed by confocal microscopy.For confocal microscopy, a Zeiss LSM410 inverted laser scanning microscope was employed. The microscope was equipped with a krypton/argon mixed-gas laser and was operated by the Microcosm Renaissance 410 (v2.3.4) software package. Images were acquired with a Plan-Apochromat 63x/1.4 oil immersion objective . Additional optical magnification (up to 5-fold) was achieved using the zoom feature of the image acquisition software. For two-color analysis, objects were excited using 488/568 nm laser lines. Green and red emissions were recorded through appropriate filters (515–540 nm band pass filter for Cy2 and 590 nm long pass filter for Texas-Red) and stored in separate (red and green) image channels. At the same time, bright field images (Nomarski optics) were collected and stored in a third (blue) channel. Image quality was enhanced during data acquisition using the Renaissance 410 line average feature (8 or 16x).None declared.S. K. carried out immunoblot analyses, infectivity assays, and was involved in the construction of plasmids and the production of antibodies. E.M., M.A.K., H.T., S.O., and R.G. participated in immunoblot analyses, infectivity studies, sample preparations, data validation, and overall experimental design. K.S. conceived of the study, performed IFA analyses, and coordinated the study. S.K. and K.S. participated in the writing of the manuscript. All authors read and approved the final manuscript.


Cervical ripening is an inflammatory reaction. The glucocorticoid receptor (GR) mediates glucocorticoid anti-inflammatory reactions, whereas nuclear factor (NF)kappaB is a key pro-inflammatory transcription factor. Prostaglandins as well as platelet activating factor (PAF) are inflammatory mediators. Inducible nitric oxide synthase (iNOS) regulates the level of nitric oxide (NO) in response to various inflammatory stimuli. We hypothesize that a changed biological response to glucocorticoids could be a mechanism regulating the inflammatory events resulting in cervical ripening.We monitored GR and NFkappaB, prostaglandin synthases cyclooxygenase (COX)-1 and -2, iNOS, as well as the PAF-receptor (PAF-R) in the uterine cervix from term pregnant women (with unripe cervices) before the onset of labor (TP), immediately after parturition (PP), as compared to non-pregnant (NP), using immunohistochemistry and RT-PCR.The GR protein was detected by immunohistochemistry in the nuclei of stroma and squamous epithelium (SQ). Stromal GR staining was increased in TP as compared to the NP group and decreased again after parturition. GR staining in SQ was decreased after parturition as compared to term. NFkappaB was present in SQ and glandular epithelium (GE), stroma and vascular endothelium. Increased nuclear NFkappaB staining was observed postpartum as compared to term pregnancy in stroma and GE. Stromal immunostaining for COX-1 as well as COX-2 was increased in the TP and PP groups as compared to the NP, and GE displayed an intensely increased COX-2 immunostaining at term and postpartum. Stromal PAF-R immunostaining was highest at term, while it was greatly increased in GE postpartum.No difference in the immunostaining for iNOS was found between the groups. RT-PCR showed a predominance of GRalpha to GRbeta mRNA in cervical tissue. The COX-2 mRNA level was increased in the PP group as compared to the TP group.There is a decrease in GR levels in human cervix at parturition. Concomitantly there is an increase of factors such as NFkappaB, PAF-R, COX-1 and COX-2, suggesting that they may participate in the sequence of events leading to the final cervical ripening. The human uterine cervix undergoes biochemical changes resulting in softening, effacement and dilatation during pregnancy and labor. This remodeling, or ripening, is a prerequisite for parturition . It is cAmong the group of structurally related, ligand-inducible nuclear steroid receptor transcription factor proteins, GR was the first that was cloned and sequenced . GR and NFκB is a key pro-inflammatory regulator. GR and NFκB are both inducible transcription factors with diametrically opposed functions in inflammatory responses. A mutual negative direct and indirect cross talk between GR and NFκB has been well described in previous studies ,14.2 (PGE2) is widely implicated for cervical ripening in clinical practice [Prostaglandin Epractice . Among tpractice . The COXpractice .Platelet-activating factor (PAF) is a lipid pro-inflammatory mediator, involved in several reproductive processes, i.e. parturition . PAF is Nitric oxide (NO) is synthesized intracellularly from the amino acid L-arginine through the activity of specific synthase enzymes (NOS) . The ind2 release from human cervical tissue explants [2 concentrations in inflammatory tissues [2 in human cervix [NO stimulates PGEexplants , and is  tissues ,25. NO d tissues ,27, at t tissues  and in n tissues . BesidesOur hypothesis is that glucocorticoids exert a direct receptor mediated effect in the human cervix uteri, and that a changed biological response to glucocorticoids could be a mechanism behind the events resulting in cervical ripening at parturition. Since NFκB has opposed functions in inflammatory responses as compared to GR, we presume that NFκB could also be a regulator of the inflammatory events leading to cervical ripening. These inflammatory events could be mediated via factors such as the PAF-R, iNOS and/or COX enzymes.All women gave their informed consent and the Local Ethics Committee of the Karolinska Hospital approved the study. All were healthy, had uncomplicated pregnancies and were without medication prior to parturition. The non-pregnant (NP) women were hysterectomised due to benign disorders not involving the cervix. The women in the term pregnant (TP) group all had unripe cervices with a Bishop score <5 points and none of them were in labor. Biopsies were obtained during elective caesarean sections before onset of labor. The biopsies from the post partal (PP) women were taken after a normal vaginal delivery.For the immunohistochemistry study of GR and NFκB, cervical biopsies were obtained from one para and eleven primipara TP women (n = 12) with a mean age (range) of 33 (28–38) years, and a mean gestational age of 38 (37 to 40) weeks. The women of the PP group (n = 14) were all primipara and had a mean age of 31 (22–37) years, and a gestational age of 40 (39 to 42) weeks. The NP control group (n = 8) had a mean age of 43 (32–49) years, and a mean parity of II (I-III).The cervical samples available for the immunohistochemistry studies of COX-1, COX-2 and PAF-R were TP (n = 8), PP (n = 10) and NP group (n = 6). Biopsies for RNA preparations were not available for the RT-PCR study from all subjects, NP (n = 5), TP (n = 6) and PP (n = 5). The women in the NP group were significantly older than those of the other two groups. Since hysterectomies in young women are uncommon most biopsies in the NP group are from women in the middle of their 40s, but they were all menstruating regularly and did not receive any medication.Cervical biopsies were obtained transvaginally (for the TP and PP groups) from the anterior cervical lip at the 12 o'clock position, from 10–20 mm depth. The tissue samples from the hysterectomies were obtained directly after the uterus was removed during operation. The same physician (YSV) collected all the samples. The biopsies were immersion-fixed in 4% phosphate buffered formaldehyde at 4°C overnight, stored at 4°C in 70% ethanol and thereafter embedded in paraffin. From the biopsies that were large enough, a small piece was cut off prior to fixation, and frozen in -70°C until RNA preparation.2, 7.5 mmol/l dithiothretiol, 0.5 mmol dNTPs, 1 μg random hexameters, and 400 U of Moloney murine leukemia virus reverse transcriptase .Total RNA from frozen cervical tissue samples was purified with the SV Total RNA isolation system  according to a procedure recommended by manufacturer. One microgram of total RNA from each sample was reverse transcribed at 42°C for 45 min in a final volume of 40 μl with a reaction mixture containing 50 mmol/l Tris-HCl (pH 8.3), 75 mmol/l KCl, 3 mmol/l MgClOligonucleotide primers for the GRα gene were as follows : 5'-CCT ® master mix  containing 2.5 μM of each oligonucleotide primer in a final volume of 20 μl. The reaction mixture was overlaid with mineral oil. After an initial incubation for 15 min at 95°C, the samples were subjected to 33  and 40 (GRβ) cycles of 30 s at 94°C, 40 s at 60°C, and 60 s at 72°C with a final extension step at 72°C for 10 min in the DNA Thermal Cycler 480 . The amount of PCR product for GRα increased linearly up to 36 cycles and for COX-1 and COX-2 it increased linearly up to 38 cycles (data not shown). Quantitative measurement of GRβ mRNA would require larger amounts of cervical RNA than were available, since GRβ showed very low expression with a visible band only after 40 cycles.For PCR, the cDNAs corresponding to 50 ng RNA were added to 10 μl of HotStarTaqTo standardize the quantification method, an endogenous 18S rRNA was used as an internal standard. The 18S rRNA primers and Competimers™ (modified at their 3' ends to block extension by DNA polymerase) were obtained from Ambion . The standard, and the GR and COX mRNAs were amplified in parallel and under the same conditions. A mixture of 18S primers and Competimers™ (1:9) was used to modulate amplification efficiency of 18S rRNA to the same linear range as GRα, COX-1 and COX-2 when amplified under the same conditions. The predicted size of the PCR product for 18S was 489 bp. The PCR products were run on 2% agarose gel and stained with ethidium bromide. Bands were captured and analyzed using ChemiDoc Gel Documentation System . The levels of GR and COX PCR products were normalized against the 18S product. The RT-PCRs were repeated twice.Paraffin sections (5 μm) were used and a standard immunohistochemical technique (avidin-biotin-peroxidase) was carried out as described before  to visuaReplacing the primary antibody with non-immune goat IgG was used as negative control.A monoclonal mouse anti-iNOS antibody was used for detection of iNOS . It recognizes the C-terminal domain of iNOS. The antibody was diluted 1:50 and incubated for 70 minutes in room temperature. Replacing the primary antibody with non-immune mouse IgG was used as negative control.A microscope and CCD video camera connected to a computer with an image analysis program  was used to assess quantitative values from GR and NFκB immunohistochemistry. The quantification of nuclear immunostaining was performed on the digitized images of systematic randomly selected fields of stroma and squamous epithelium. Ten fields were analyzed separately in each section of tissue, using the color-discrimination software. Positive staining is presented as a ratio of the area of positively stained nuclei (brown) to the total area of cell nuclei (brown and blue).Two observers blinded to the identity of the slides, performed all the assessments. The staining was evaluated semi-quantitatively using a grading system. The staining intensity was graded on a scale of (0) no staining, (1) very faint, (2) faint, (3) moderate and (4) strong staining.Statistical calculations for the data from the relative quantification of RT-PCR products, the immunohistochemistry results by image analysis and manual scoring were performed by ANOVA on Ranks  and significances were evaluated by Dunn's test. Values with different letter designations are significantly different (p < 0.05).By immunohistochemistry the GR protein was localized to the nuclei of cervical stroma (S), squamous epithelium (SQ), glandular epithelium (GE) and vascular endothelium Figure  in samplPositive NFκB immunostaining in cervix uteri was present in stroma, GE, SQ and vascular endothelium in NP (left column), TP (middle column) and PP (right column) groups Figure . Strong Immunostaining for COX-1 Figure  was founImmunostaining of COX-2 Figure  was founImmunostaining of PAF-R was found in stroma, neuronal ganglion (G) and GE Figure . The immFaint positive staining was found in SQ, stroma and GE Figure . There wThe GRα mRNA level was determined by RT-PCR Figure . GRα mRNVery low levels of GRβ mRNA were present in 4 out of 6 samples from the TP group, and in 2 out of 5 samples in the NP and PP groups Figure .There was no difference in the COX-1 mRNA level between the groups, as assessed by RT-PCR Figure  Figure , top.The COX-2 mRNA level was the highest in the PP group Figure  Figure , bottom,This study is, to our knowledge, the first reporting GR expression in human cervix uteri during pregnancy and at parturition. In a recent study on human endometrium , GR protGlucocorticoids exert their anti-inflammatory effects primarily by inhibiting the expression of cytokines e.g. IL-8, as demonstrated in human fibroblasts , tumour Maternal plasma levels of cortisol increase until term , but theOur data shows that the GR protein was present in cervical stroma, SQ and vascular endothelium in samples from non-pregnant, term pregnant and postpartum women. The decrease in GR levels in stroma and SQ at parturition, as compared to term, could be interpreted in terms of a GR mediated glucocorticoid anti-inflammatory activity during pregnancy that is ended at parturition.The NFκB protein was present in cervices from non-pregnant, term pregnant and postpartum women. This protein, ubiquitously expressed in a variety of cell types, is found in the cytoplasm in its inactive form, but translocates into the nucleus upon activation . TherefoPolymorphonuclear leukocytes and macrophages migrate from blood vessels and accumulate in the cervix uteri before parturition . GR and 2 in the human body [Cortisol is a potent inhibitor of COX-2 in myometrium, decidua and cervix , and ref. This woman body . We coulman body , but alsman body , and refman body , the act2 release from human cervical tissue explants [2 in human uterine cervix [Nitric oxide stimulates PGEexplants . Nitric explants . Treatmee cervix . The imme cervix ,48. Tsche cervix . LedinghPlatelet activating factor (PAF), like prostaglandins derived from the arachidonic acid precursor, is a multifactorial pro-inflammatory mediator, which has been implicated in parturition. Local application of PAF in rats induced cervical ripening , whereasIf the process of cervical ripening is disturbed, either resulting in a preterm delivery or to a prolonged delivery time, possibly ended by a cesarean section, it will lead to increased risks for both the mother and the child. Preterm delivery is the leading factor causing neonatal mortality and morbidity . An incrWe have demonstrated that the human uterine cervix is a potential target organ for glucocorticoids during pregnancy. The higher GR protein levels in cervical stroma and SQ before parturition may reflect a GR mediated anti-inflammatory effect of cortisol during pregnancy, with a subsequent decline of this activity at parturition. The concomitant increase in nuclear NFκB levels in the cervix suggests activation of this transcription factor at parturition. NFκB activity promotes pro-inflammatory events and could be responsible, at least in part, for the observed increase in COX-1, COX-2 and PAF-R levels.


Current methods for accurate quantification of nucleic acids typically begin with a template preparation step in which DNA and/or RNA are freed of bound proteins and are then purified. Isolation of RNA is particularly challenging because this molecule is sensitive to elevated temperatures and is degraded by RNases, which therefore have to be immediately inactivated upon cell lysis. Many protocols for nucleic acids purification, reverse transcription of RNA and/or amplification of DNA require repeated transfers from tube to tube and other manipulations during which materials may be lost.Xist, Sry and the two heat-inducible hsp70i (hsp70.1 and hsp70.3), as well as their RNA transcripts in single mouse embryos and in isolated blastomeres.This paper introduces a novel and highly reliable single-tube method for rapid cell lysis, followed by quantitative preparation and analysis of both RNA and/or DNA molecules in small samples. In contrast to previous approaches, this procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction or binding to a matrix. We demonstrate the utility of this method by quantification of four genes, This method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules. Real-time polymerase chain reaction (PCR) in combination with reverse transcription (RT) provides a powerful tool for accurate quantification of DNA and RNA copy numbers and has opened the way to the study of subtle modulations of gene expression in small numbers of cells, as well as small-scale genetic analyses aimed at establishing chromosome numbers, the presence of mutations, or allele dropout. The reliability of these measurements, however, depends on the accuracy of each step, including preparation and recovery of RNA and/or DNA, reverse transcription of RNA into cDNA, and quantifiable and specific amplification of all desired sequences. The importance of optimizing each of these steps is well recognized , as is tOur laboratory has already demonstrated that bound proteins prevent reliable PCR amplification of genomic DNA and that a thorough proteolytic digestion followed by heat inactivation solves this problem . For accThe chaotropic agent guanidine isothiocyanate (GITC) has long been the chemical of choice for nucleic acid preparation. It is particularly useful for RNA studies ,9, becauIn view of these limitations we devised an alternative strategy in which the sample is collected and denatured in a minimal volume of a GITC solution, briefly heated to allow dry storage, and, when needed, is directly analyzed in the same tube by performing quantitative RT-PCR in a volume large enough to lower GITC concentration to negligible levels. This new procedure, hereafter referred to as PurAmp (patent pending) and described here in full for the first time, is the only available method that allows a whole sample, such as a single embryo or cell, to be processed from lysis to RT-PCR in the same tube, under conditions that permit precise quantification of both RNA and genomic DNA copy numbers. This fully optimized method is sensitive enough to detect specific sequences within a single chromosome in one cell, yet robust enough to measure the presence of thousands of RNA molecules released from hundreds of cells.Xist, responsible for X-inactivation and dosage compensation in female cells [hsp70.1 and hsp70.3, jointly called hsp70i [Xist RNA is a noncoding transcript that exerts its particular function of gene-silencer by coating the inactive X-chromosome. Unlike hsp70i RNA and most other mRNAs, it is therefore localized in the cell nucleus and particularly challenging to extract. Besides their biological relevance, both Xist and the hsp70i genes offer the advantage of naturally-occurring unambiguous controls for the specificity of transcripts amplified with RT-PCR. Xist RNA is, in fact, virtually absent from male cleavage stage embryos [hsp70i RNA is predominantly synthesized in response to stress although minimal levels of hsp70i transcripts are normally present in embryonic cells. A careful quantitative analysis of hsp70i heat and culture stress-response in preimplantation embryos at different developmental stages, and its implications for development, will be presented elsewhere . In addition, we have been able to detect and quantify the genomic sequences of Xist, the hsp70i and the male sex-determining gene Sry [PurAmp has made it possible for us to conveniently investigate expression levels of two gene types essential for early mouse embryo development: le cells -13, and d hsp70i ,15. Xist embryos -19, whilgene Sry . BecauseThe PurAmp method presented in this study is performed in a single tube from cell lysis to cDNA or genomic DNA amplification, thus eliminating possible loss of template molecules due to procedures such as phase separation and recovery, repeated washing and re-suspension of nucleic acid pellets, elution from binding matrices and vessel-to-vessel transfer. This strategy offers an immediate improvement in the precision of gene expression analyses, at the same time shortening considerably the experimental protocol compared to traditional methods.Xist RNA content of a group of female mouse embryos at the blastocysts stage and quantified Xist and Sry DNA copy numbers in their male counterparts. Our previous analysis of these parameters in single female and male mouse embryos at different developmental stages provided us with an ample pool of data obtained with commercially available nucleic acids preparation methods [In order to validate our method, we initially measured the  methods -19, whicXist/Sry amplicons was detected using two molecular beacons conjugated to different fluorescent dyes. Both amplicons span intronless sequences of the genes [Sry and Xist signal. Three embryos were identified as male based on the presence of the Sry amplicon  as the Sry signal . This indicates the presence of the same number of copies of Sry and Xist templates, as expected for male blastomeres that contain one copy of the Sry gene on the Y-chromosome and one copy of the Xist gene on the X-chromosome. Neither gene is expressed in male blastocysts [Xist and Sry DNA. The three remaining embryos did not generate any Sry signal , the accumulation of Xist RNA per female blastocyst averages above 12,000 copies, considerably higher than our previous measurement of 6797 ± 2894 copies obtained using a multistep nucleic acids isolation procedure [Xist and Sry sequences is neither decreased nor increased by the presence of diluted denaturing solution  during real-time PCR. In fact, additional experiments revealed that Xist/Sry real-time PCR was unaffected by a GITC concentration as high as 20 mM (not shown). Taken together these results suggest that the higher levels of Xist cDNA measured using the PurAmp method are due to an improvement in RNA recovery at the initial step of cell lysis.Quantification of the real-time PCR data obtained from the three male embryos in Fig. astocyst . Both vaastocyst , a phenorocedure . The datXist and Sry genes and thousands of copies of Xist transcripts, with rather wide sample-to-sample fluctuations. In order to more carefully determine the quantitative capability of the new assay, we next analyzed embryos at earlier developmental stages containing lower and, in some cases, precisely known numbers of template copies. Figure Xist amplicons generated in the course of two separate experiments by, right-to-left, i) a 3-cell male embryo (yellow); ii) a 4-cell male embryo (green); iii) a single blastomere isolated from a 4-cell female embryo (light purple); iiii) a 4-cell female embryo (red); iiiii) a female blastocyst (blue). The gender of each embryo was confirmed by the detection of an Sry-specific fluorescent signal in male samples , denoting the presence of Xist transcripts , albeit at considerably lower levels than those measured in the female blastocyst . These measurements are consistent with other studies demonstrating that Xist transcripts are accumulated in the developing embryo beginning at the late 2-cell stage [Xist developmental profile [As shown above, blastocysts are comprised by many cells and contain hundreds of copies of the ther two , an obsele cells ,17. Convll stage  and withXist signal generated by a single blastomere isolated from a 4-cell female embryo arose 2.2 cycles later than the Xist signal of the whole 4-cell female embryo  RNAs.In order to more extensively test the validity of the PurAmp approach to template quantification in single cells, we measured transcript levels of the heat shock-inducible genes  protein ,15. For hsp70i expression. Like Sry, the hsp70i are naturally intronless genes and therefore once again our pair of PCR primers simultaneously amplified both genomic DNA and cDNA sequences. The data in Table hsp70i template numbers due to the presence of thousands of copies of hsp70i RNA, although these numbers were considerably lower when samples were prepared with a multistep/multitube phenol-chloroform extraction [hsp70i RNA levels. Based on these results, the duration of the recovery period was increased to at least two hours in all following experiments, eliminating the finding of "non responsive" embryos.A preliminary set of experiments was carried out on embryos at the blastocyst stage, when heat shock response is fully established , with thtraction ,18 rathehsp70i RNA were then quantified in whole 8-cell embryos that had or had not been exposed to a temperature increase, as summarized in Table hsp70i genomic DNA copies measured in the absence of RT was consistent with the presence of four copies of the genes per cell, one hsp70.1 and one hsp70.3 on each chromosome 17, and with the fact that some of the cells analyzed had already duplicated their DNA. Only a minimal amount of hsp70i RNA, calculated as the difference of template copy numbers obtained with and without reverse transcription (hsp70i cDNA + genomic DNA less hsp70i genomic DNA), was present in non-heated embryos, indicating that the embryos were not stressed by culture conditions [Copy numbers of nditions .hsp70i RNA increased sharply after a 30-minute heat treatment, followed by a recovery period of either 2 or 3 hours necessary for transcripts synthesis and accumulation. Some of the heat-shocked embryos were harvested intact, while others were dissociated in single blastomeres. The average numbers of hsp70i template copies per blastomere were calculated from the isolated cells  and compared to the average per blastomere values obtained from whole embryos. The results show that the amount of hsp70i RNA + genomic DNA per cell calculated by these two approaches is very similar. A post-heating recovery period of 3 hours rather than 2 hours increased the hsp70i RNA levels only slightly, indicating that the onset of transcription and the major build-up in RNA occur quickly. Single cells derived from non-heated embryos contained only trace amounts of hsp70i RNA, consistent with whole embryo measurements, and PCR efficiency was, again, unaffected by the PurAmp components (not shown).Expression of Xist/Sry PurAmp protocol and analyzed the efficacy with which the genomic DNA was degraded. Genome numbers in embryos at the morula stage were calculated by counting Xist and Sry copies in five male samples  and averaged at 21.3 ± 8.9, consistent with the fact that embryos at this stage are normally comprised of 16-to-32 cells. After treatment with DNase I, only 0.8 ± 1.5 genomes per embryo were still present in a group of 15 male samples, demonstrating that the enzyme had successfully degraded 96.3% of the DNA. Figure Xist (upper panel) and Sry (lower panel) DNA in a group of eight male and eight female single embryos. As expected, control male samples (blue lines) contained equal numbers of Xist and Sry copies, as shown by the equal CT values, corresponding to the genomic DNA copy number. Both amplicons were absent from DNase-treated embryos that could be identified as male because they were devoid of Xist RNA (yellow lines). In contrast, control female samples (red lines) contained Xist RNA and DNA but lacked Sry. DNase treatment caused a delay in the Xist signals arising from female embryos (green lines), consistent with the elimination of all 42 copies of genomic Xist DNA in each embryo plus some decrease in the number of Xist transcripts. The amount of RNA recovered in these samples averaged at 75% of control levels. The fact that some RNA is lost during the DNase step is not surprising as it is well known that some RNA hydrolysis is unavoidable (see Discussion), and is not linked to the single-tube procedure. We anticipate that further optimization of the available DNase protocols and reagents will minimize this problem. Thus, the data in Fig. The PurAmp method described above automatically results in the quantitative recovery of genomic DNA, which can then be used as a quality control and a convenient internal standard for the simultaneous recovery and measurement of mRNA -19, see , see 19,It is increasingly clear that individual cells in a population do not exhibit identical patterns of gene expression and, hence, that expression profiling is more informative if it is quantitative and carried out at the single cell level -27. ThisXist RNA, are freed and made available to reverse transcription as well as cytoplasmic mRNA molecules, a result unattainable by mild detergent treatment that leaves nuclei intact [First, cell lysis and protein denaturation occur very rapidly upon delivery of the sample to crystalline GITC, thus ensuring both protein removal from DNA and RNA and inactivation of cellular nuclease that would otherwise quickly degrade RNA . Transcri intact . Second,Xist RNA levels in developing mouse embryos highlight one of several merits of the PurAmp method over the traditional, multistep approach to nucleic acids purification [Xist RNA levels are higher with the single-tube protocol. The same culture conditions and procedures were used in the two groups of experiments, making it unlikely that differences in embryo quality were the cause of the increase in Xist RNA. We, therefore, conclude that the higher levels of Xist RNA observed using the new procedure reflect improved template preparation with efficient inactivation of RNases and reduced loss of RNA molecules. Xist RNA is known to trigger X-chromosome silencing through interactions with numerous proteins and possibly with the nuclear matrix scaffold [hsp70i templates in heat-shocked blastocysts supports the view that larger pools of RNA are detected in PurAmp-treated samples than when using phase separation-based nucleic acid extraction. The later method, in fact, presents several steps that require extreme care to avoid loss of material, including complete recovery of the upper phase, thorough precipitation of all nucleic acids molecules, and repeated re-suspension and washing of barely visible pellets. All these manipulations render the results obtained with this technique particularly operator-dependent, while, in contrast, PurAmp simply requires sequential addition of reagents into the same tube. Once the sample is delivered to the LysoDot in the reaction vessel  were obtained from Embryotech Laboratories, Inc. , and were cultured as previously described  until thXist measurements) or pre-compaction 8-cell embryos (for hsp70i measurements) after drilling the zona using a ZILOS-tk™ zona infrared laser optical system (beam = 1480 nm) , according to a procedure developed in our laboratory and described elsewhere [Single blastomeres were isolated from either 4-cell embryos  to the inside surface of the lids of PCR-grade reaction tubes . Precise measurement of the droplets size was obtained following the method previously described by Wangh . The denImmediately before harvesting, individual embryos were placed in 3 ml of Dulbecco's PBS devoid of calcium and magnesium chloride . DulbeccIn order to perform reverse transcription, each sample was carefully re-solubilized in the lid by addition of 6 μl of Random Hexamers mixture (4.2 ng/μl) in DEPC-treated water . Tubes were closed, inverted, briefly centrifuged and incubated for 5 minutes at 65°C in order to allow primer/RNA hybridization. The remaining reagents needed for RT were then added to the tube in a volume of 4 μl, and the reaction was carried out according to the protocol suggested by the manufacturer. As previously described -19, all Xist and Sry genomic DNA + cDNA templates was thus performed in a final volume of 100 μl, as detailed elsewhere, in the presence of 4 units of Taq DNA polymerase  [® 7700 Sequence Detector  and fluorescence readings were taken at the annealing temperature.The full volume of each sample was then mixed with 89.5 μl of complete PCR amplification cocktail containing sequence-specific molecular beacons as detection probes . Multiplson, WI) ,19. Realhsp70i assay was carried out similarly to the one for the Xist/Sry multiplex, by sequential dilutions of denaturant, RT and PCR reagents. The procedure for collection and lysis of the samples was the same. Dry samples were re-solubilized with 6 μl of random decamer primers (8.3 μM) in nuclease-free water . After a 3 minute incubation at 75°C to optimize primer binding to RNA, all other reagents needed for RT were added to the sample and the reaction was carried out according to the manufacturer's instructions. As for the Xist/Sry assay, all RT mixture components were used at the suggested concentrations, but volumes were halved so that RT was performed in a final volume of 10 μl. An RNase H digestion step was included at the end of RT, as in the case of the Xist/Sry assay.Embryos were heat-shocked at 43°C for 30 minutes, followed by a recovery period of 30–40 minutes (blastocysts), or 2–3 hours  at 37°C. The hsp70i primers were localized at positions 1245/1305 of the hsp70.1 GenBank sequence with accession number M35021  and were identical to sequences within the hsp70.3 gene, previously known as hsp70A1 (GenBank sequence with accession number M76613) [hsp70i was the only amplicon generated in this assay, it was not necessary to design a sequence-specific detection probe. In this case, SYBR® Green, a fluorescent dye that binds to double-stranded DNA, was used as fluorescent probe for real-time PCR. The specificity and purity of the amplicon was confirmed by both gel electrophoresis and analysis of the melt profile, as previously described [hsp70i PCR was the following: 50 mM Tris, pH 8.3, 3 mM MgCl2, 0.3 μM each primer, 0.25 mM each dNTP, 1:62,500 SYBR Green , and 4 units of Taq DNA polymerase . The polymerase was incubated at a 1:1 (v/v) ratio with Platinum® Taq antibody (Invitrogen) for 5 minutes before addition to the reaction mixture (hotstart PCR). The cycling profile was: 95°C for 5 minutes; 10 cycles consisting of the following four steps: 95°C (20 sec), 64°C (30 sec), 72°C (30 sec), 84°C (15 sec); 35 cycles with the following four steps: 95°C (20 sec), 59°C (30 sec), 72°C (30 sec), 84°C (15 sec). Fluorescence readings were acquired at 84°C, in order to exclude fluorescent signals due to the possible formation of primer dimers late in the reaction.Real-time PCR was carried out in a final volume of 100 μl, by adding the PCR reagents to the sample after completion of RNase H digestion. The chosen  M76613) . Becauseescribed . The comhsp70i genomic DNA copy numbers in the absence of cDNA [A number of embryos were also processed as "No RT" controls, with the same protocol used for the other samples but without inclusion of reverse transcriptase in the RT mixture. These controls were used to quantify  of cDNA .hsp70i expression in blastocysts, some of the embryos were processed using a commercially available multistep/multitube kit, as previously described [For the preliminary studies on escribed . BrieflyT) at which each fluorescent signal was first detected above background. CT values were compared to standard scales obtained from analysis of male mouse genomes at known copy numbers, as detailed previously [T determinations. A lower CT value indicates an earlier detection of the fluorescent signal and therefore more templates present at the start of the reaction [reviewed in ref. [Sry (Y-chromosome), one copy of Xist (X-chromosome) and four copies of hsp70i .Calculation of template copy numbers was based on the "threshold cycle"  and 1 unit of DNase I (Ambion) in nuclease-free water. After an incubation of 20 minutes at room temperature, the reaction was terminated by adding 1 μl of a 10 mM EDTA solution, pH 8.0 (Invitrogen). The nuclease was inactivated by heating the samples at 65°C for 10 minutes, according to the protocol recommended by Invitrogen. One μl of a 25 ng/μl RT primer solution was then added to the sample, so that the final primer concentration was now the same as in the assay without DNase (see above). RT and PCR were then carried out as detailed for the "No DNase" assay.Embryos were grown to the morula stage and were individually harvested and lysed as detailed above. Controls were processed for multiplex detection of Xist/Sry measurements and drafted the manuscript. AA established heat shock conditions and performed the hsp70i quantification experiments. LJW coordinated the study and contributed to all aspects of its design.CH devised the finalized form of the PurAmp method, carried out the


Although modest improvements in the survival of patients with non-small cell lung cancer (NSCLC) can be achieved with cisplatin-based chemotherapy (CT), its value is disputed in the geriatric setting. In this study, we evaluate the feasibility of vinorelbine/cisplatin CT for elderly NSCLC patients.2, on day 1 and 8, and cisplatin on day 1, in 28 days-cycles. After stratification for age (up to 75 years), younger patients were sequentially allocated to moderate cisplatin doses (80 mg/m2 or 90 mg/m2), and older patients were allocated to lower cisplatin doses (60 mg/m2 or 70 mg/m2). We recruited patients aged over 70 years with newly diagnosed NSCLC, clinical stage III or IV, Karnofsky performance status ≥ 70%, normal serum creatinine, peripheral neuropathy ≤ grade 1, and no prior cancer therapy.In this pilot phase I/II trial, all patients received CT with vinorelbine 25 mg/mAnalysis was by intention to treat. Main toxicities (grade 3–4) was as follows: neutropenia, 20%; anemia, 11%; and thrombocytopenia, 2%; alopecia, 55%; fatigue, 11%; and peripheral neurotoxicity, 2%. No grade 3–4 emesis or renal toxicity occurred. Global median time to progression (TTP) and overall survival (OS) were 27.0 (95% CI: 10.1 to 43.7) weeks and 30.1 (95% CI: 24.4 to 35.8) weeks; 1- and 2-year survival rates were 36.3% and 13.2%, respectively. Overall response rate was 50.0% (95% CI: 35.4% to 64.5%), with 1 complete response; no difference on response rate was noticed according to cisplatin dose. Median overall survival was 30.1 weeks, with 1- and 2-year survival rates of 36.3% and 13.2%, respectively.Age does not preclude assessment on the role of cisplatin-vinorelbine CT for elderly NSCLC patients with good performance status and adequate bodily functions. Lung cancer is a disease with a great incidence in older people. In Brazil, its incidence rate in male sex was expected to reach 18.8:100.000 in 1999, and aged patients may have accounted for 57% of all lung cancer deaths. Unfortunately, up to two thirds will present at diagnosis with advanced disease, requiring chemotherapy (CT) [Vinorelbine, a semi synthetic vinca alkaloid, is a highly active drug for NSCLC and its association with cisplatin is worthwhile. European and Southwest Oncology Group trials demonstrated that vinorelbine/cisplatin (VP) offer therapeutic advantage over both drugs alone -4, previAlthough modest improvements in the survival of patients with non-small cell lung cancer (NSCLC) can be achieved with cisplatin-based CT , its val9/L and platelets ≥ 100 × 109/L), bilirubin under 1.25 times upper normal value (UNV), aspartate aminotransferase/alanine aminotransferase (AST/ALT) under 2 times UNV, and renal function (creatinine level under 120 μmol/L); no symptomatic brain metastasis; no prior cancer therapy; no indication for palliative radiation therapy; no previous or concomitant malignancy; and adequate social support. Exclusion criteria were symptomatic peripheral neuropathy and comorbidity regarded as an impediment for CT, such as renal disease, heart failure, coronary heart disease, uncontrolled infection, and cognitive impairment.This study enrolled patients older than 70 years with unresectable locally advanced or metastatic NSCLC. Informed consent was obtained from patients and their relatives, as approved by the Institutional Ethical Committee. Inclusion criteria: all patients had to have histologically confirmed NSCLC; Karnofsky performance status ≥ 70; measurable disease; adequate bone marrow reserve  and biochemistry; chest x-ray; bone scintigraphy scan; chest, abdominal and brain computed axial tomography scans; and electrocardiogram. Although pretreatment bone scan and brain CAT scan are recommended only when signs or symptoms of disease are present, they were added here due to Institutional Protocol Reviewers recommendation.2 on days 1 and 8, administered intravenously in bolus, followed by intravenous cisplatin over 1 hour. CT was administered every 4 weeks. Prophylactic anti-emetic drugs (intra venous dexamethasone 20 mg and ondansetron 16 mg) and fluid hydration  was used to minimize renal toxicity. Dexamethasone 4 mg PO BID plus metoclopramide 10 mg PO QID for 4 days was used to prevent delayed emesis. Patients were time-sequentially assigned to one of two groups, from lower to higher doses, according to age strata. Study doses were defined based on previous reports on renal tolerance of cisplatin in geriatric patients [2 and those aged 70 to 75 received cisplatin 80 or 90 mg/m2. Assignation to high dose groups (70 mg/m2 and 90 mg/m2) occurred after evaluation of toxicity at inferior doses, evaluated according to National Cancer Institute criteria. The protocol required a minimum of 6 and a maximum of 18 patients per group after the 1st cycle for safety analysis.Treatment consisted of vinorelbine 25 mg/mpatients . Age str9/L and platelets were <100 × 109/L, treatment was delayed by 1 week. Treament on days 8 had to be cancelled if neutrophil counts were <1.0 × 109/L and platelets were <100 × 109/L. If treatment could not be given after a 2-week interval because of haematological toxicity, it had to be discontinued and the patient withdrawn from the study. Concomitant use of hematopoietic growth factors were not allowed in the first treatment cycle but were administered subsequently on individual basis. Neurological toxicity above grade 2 resulted in suspension of treatment; ototoxicity grade 2 or 3 resulted in a 50% dose reduction of cisplatin.Chemotherapy doses were reduced for haematological, neurological, hepatic and renal toxicities. Toxicity was graded according to National Cancer Institute (NCI) common toxicity criteria guidelines. Changes in dosage were based on WBC results obtained on day 1 of treatment; if neutrophils were <1.5 × 10The following dose modifications of vinorelbine were set based on AST/ALT  and bilirubin values on day 1 or day 8 of treatment: if AST/ALT were between 5.1 and 20.0 × UNV or bilirubin was between 1.5 and 3.0 × UNV, dosing was cancelled and the patient was reassessed 1 week later. If AST/ALT were >20.0 × UNV or bilirubin was >3.0 × UNV, vinorelbine was discontinued. If serum creatinine was grade >1, the dose was delayed by 1 week and the test repeated. After a 2-week delay, the patient was taken off the study. WBC and biochemistry were also performed on day + 14 of treatment.We intended to administer a maximum of four CT cycles followed by radiation therapy in responding patients with stage III disease and six CT cycles in patients with wet IIIB or stage IV disease. Notwithstanding radical radiation therapy (RT) should deliver a total dose down to 66 Gy, covering tumor site and regional lymph nodes, and palliative therapy could use doses under to 45 Gy, the final choice of dose, fractioning, irradiated volume, and energies of radiation was at the radiation oncologist's discretion. Treatment interruption was allowed in case of disease progression, severe adverse events, or patient preference.Chest x-ray was performed before each cycle, and CAT scans every two cycle for response evaluation. Tumor response was recorded according to World Health Organization criteria and measured by the same observer (JRP). All responses had to be confirmed 3–4 weeks from initial evaluation. We reported here the best response designation recorded from the start of treatment until disease progression. Patients stopping treatment with an unconfirmed response, or only short stabilisation were considered as inevaluable, unless the response or stabilisation was further confirmed in the absence of any treatment. Patients were monitored for the first month off-study then followed up every 2–3 months.The dose intensity was calculated for both drugs by dividing the actual dose delivered by the length of therapy. Toxic death was defined as death occurring during the chemotherapeutic phase (including four weeks after its end) and due to drug toxicity. Early death was defined as death within four weeks after a chemotherapy cycle without severe toxicity and not related to the malignant disease. Response and survival were calculated by intention-to-treat. Progression was defined in relation to the best response obtained. The time to tumor progression lasted from the first day of treatment to the date of the first observation of progressive disease. Survival was defined as the time elapsed from the beginning of CT until death or last follow-up visit. Time-to-event analysis was performed using the Kaplan-Meier product-limit estimator. All analyses were carried out using a computer program .2 and eight received 70 mg/m2; in the former, fifteen patients received 80 mg/m2 and fourteen received 90 mg/m2. Patient characteristics are given in Table Forty-four patients were recruited from July 1996 to June 1998; twenty-nine aged 70–75 year and fifteen older than 75 years. Cisplatin doses were as follows: in the older cohort, seven patients received 60 mg/mTreatment results are shown in Table Toxicity data are presented in Table 2 on day 1 plus vinorelbine 25 mg/m2 on days 1 and 8, repeated each 28 days per in the maximum four cycles, was feasible for elderly NSCLC patients. Neurological and renal tolerance was particularly good. At the time we developed the protocol, no quality-of-life instrument had been validated for use in Brazil. Thus, a drawback in our study is the absence of quality-of-life analysis, which precludes evaluation of key dimensions in geriatric oncology.Treatment of elderly NSCLC patients with cisplatin-based regimens has been a less contentious matter nowadays but toxicity remains a major issue. In our pilot study, chemotherapy with cisplatin 70–80 mg/mCisplatin induces a sensory neuropathy due to axonal damage that is dependent on the total-dose and single-dose intensity , and it Cisplatin renal toxicity has been attributed to drug-protein interactions and the inactivation of specific brush border enzymes, resulting in damage of the loops of Henle, the distal tubules, and collecting ducts. Patients aged above 70 or even 80 are regarded as susceptible to cisplatin-induced renal damage as the younger counterparts ,16 and cThe observed response rate here was in the usually range of NSCLC phase II trials, but the absence of external review of radiological data and the widened confidence intervals expected because of small sample sizes in each group limit assertions that could otherwise be drawn. The 1-year survival rate (36%) was good but essentially equivalent to the reported elsewhere  for vinoVinorelbine is a cytotoxic agent that clearly has expanded the therapeutic options for elderly NSCLC patients ,21. The Although sequential administration of drugs is an attractive option for aged or frail patients, a setting where minimal treatment-related toxicity should be pursued, research on the role of non-platinum combinations for elderly NSCLC patients aroused attention. The Southern Italy Cooperative Oncology Group (SICOG) evaluated whether the association of vinorelbine and gemcitabine would be better than vinorelbine alone. To date, final results of this trial have not to come. Despite an article focusing on the interim analysis of 120 patients (60 at each group) claimed a survival advantage for the combined arm , furtherThe question whether or not cisplatin-containing regimens should be used to treat aged patients remains an important, still open, issue. As observed for paclitaxel-carboplatin , gemcitaThis study was supported by Institutional funds only. Authors did hot received reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this paper in the past five years.NSCLC: non-small cell lung cancerCT: chemotherapyNCI: National Cancer InstituteELVIS: Elderly Lung Cancer Vinorelbine Italian StudySICOG: Southern Italy Cooperative Oncology GroupGFR: glomerular filtration rateJRP was responsible for study conception, protocol conduction, and results interpretation. SJM carried out the data analysis and results discussion. SMN and FKI were responsible for patient care and data gatheringThe pre-publication history for this paper can be accessed here:


Medical ethics has existed since the time of Hippocrates. However, formal training in bioethics did not become established until a few decades ago. Bioethics has gained a strong foothold in health sciences in the developed world, especially in Europe and North America. The situation is quite different in many developing countries. In most African countries, bioethics – as established and practiced today in the west- is either non-existent or is rudimentary.Though bioethics has come of age in the developed and some developing countries, it is still largely "foreign" to most African countries. In some parts of Africa, some bioethics conferences have been held in the past decade to create research ethics awareness and ensure conformity to international guidelines for research with human participants. This idea has arisen in recognition of the genuine need to develop capacity for reviewing the ethics of research in Africa. It is also a condition required by external sponsors of collaborative research in Africa. The awareness and interest that these conferences have aroused need to be further strengthened and extended beyond research ethics to clinical practice. By and large, bioethics education in schools that train doctors and other health care providers is the hook that anchors both research ethics and clinical ethics.This communication reviews the current situation of bioethics in Africa as it applies to research ethics workshops and proposes that in spite of the present efforts to integrate ethics into biomedical research in Africa, much still needs to be done to accomplish this. A more comprehensive approach to bioethics with an all-inclusive benefit is to incorporate formal ethics education into health training institutions in Africa. Medical Ethics, which became the template on which the code of ethics of the American Medical Association was based in 1847 [Before the modern discipline of bioethics evolved, ethics had been on the centre stage of medical practice for more than two millennia, since the time of Hippocrates. In 1803, Thomas Percival published his book on  in 1847 . Of the The scope of bioethics has continued to expand in response to changes in societal dynamics, medical technology and health care practices. Perhaps more because of its antecedents than for want of content, most of the discourse and writings in the early days of bioethics centred on issues of patient-physician relationship, respect for person, best interest of the patient, and justice in health care delivery. Today, traditional bioethics discussions and literature are changing as new ethical concerns evolve around dilemmas posed by new technology on the subjects of end of life issues, organ donation, human reproduction and human genomics. Moreover, the bioethics agenda has expanded to include the subjects of resource allocation, organizational ethics and public health ethics, among others.Bioethics in its present form is rooted in and largely dominated by western culture. The tempo and content of bioethics discourse are largely influenced by the technological creations of the developed world. However, ethics is not exclusively the domain of the west. Core ethical values are essentially the same for all human communities leaving aside each community's customs, culture and preferences . AccordiIn the bioethics literature, bioethical discourse and arguments have been most prominent and intense concerning research involving human participants. One major achievement in this regard is the creation of an oversight body that sees to the proper design and conduct of research that conforms to generally acceptable and established ethical guidelines. There resides in this body the duty of ensuring that research sponsors and investigators abide by established conventions for carrying out clinical research. They also perform the role of assuring the safety of research participants and ensuring that participants (and/or society) benefit from the outcome of research. The relevance of this body to modern day health care and research is partly exemplified in the absence of any major scandals in the form and magnitude of those already recorded in history. The establishment of research ethics boards has not solved all the ethical problems of biomedical research though. There is still a lot to do to re-structure, re-empower and re-position the board to match the complexities of the present-day technology-driven medical research and practice.Various bodies within and outside Africa have pioneered the movement towards ensuring that medical research in Africa conforms to international ethical guidelines. This is the aspect of bioethics that is most visible in Africa and has been anchored partly by the Pan African Bioethics Initiative (PABIN), a pan-African organization that was established in 2001 to foster the development of bioethics in Africa with a particular focus on research ethics (5). This idea has arisen in recognition of the genuine need to develop capacity for reviewing the ethics of research in Africa. It is also a condition required by external sponsors of collaborative research in Africa. Ethics workshops and conferences have been held in different parts of Africa, including Tanzania , Zambia While the present efforts and achievements are commendable, much still needs to be done for the effects to filter through to the grassroots, which is the main arena of research activities and where the burdens of research are most felt. I say this for the following reasons. First, the present efforts are still limited in extent and effect. Hitherto, most of the conferences have taken place in two or three geographical zones of the continent and have been limited to a few days of activities. Of course, the interests and motives of the sponsoring agencies together with the presence on the ground of those who are available to organize the conference locally determine where, when, for how long and the number of participants in the workshop. There is thus restriction on the number of researchers who can attend the conferences from all over Africa and on the amount of knowledge that can be imbibed in those few days. The consequence is that the same few people attend the conferences most of the time. These attendees from a few centres might not be able to change unethical research practices in their countries. Attempts by these few to train their colleagues locally are often constrained by lack of funds.Second, absence of national directories of research activities in most African countries makes the magnitude of biomedical and social sciences research in Africa to be underestimated. For example in Nigeria, five categories of research and researchers are easily identifiable. Individual or institution-supported research is done by students, clinicians (including resident doctors) and other scientists. This category constitutes a significant proportion of research in tertiary academic and health institutions. Industry-sponsored research is undertaken by researchers for pharmaceutical companies to promote new or old drugs. Such research protocol may be indigenously developed or be a part of multicentre trials. In most instances, these companies do not go through the institutions where the researchers are based, but deal directly with individual researchers, who may or may not subject the research protocol to an ethics board review. Collaborative research with colleagues from the developed countries is often externally funded. It includes hospital and community-based trials and mostly involves experimenting with drugs or vaccines. Of particular ethical concern in collaborative research is the fact that external sponsors may differ in their motives for conducting research and there may be limited applicability of research benefits to the country or local community . MoreoveThird, a majority of Africa's research participants are highly vulnerable given their low level of formal education and the political, social and economic milieu in which they live. The fourth reason is that Africa is a pluralistic society with diverse peoples and cultures. While general guidelines may apply in most cases, in some the peculiarities of each ethnic and cultural group will significantly affect what research is done and how it is done in those communities. Lastly, not every research centre has established an ethics review process. Where already established, most of the ethics review committees are grossly underfunded and unequipped for their duties.This is the branch of bioethics that addresses ethical conflicts that arise in daily clinical practice in health care institutions, through the establishment of hospital ethics committees and ethics consultation services. Fletcher and Siegler define ethics consultation "as a service provided by an individual consultant, team or committee to address the ethical issues involved in a specific clinical case. Its central purpose is to improve the process and outcomes of patents' care by helping to identify, analyze, and resolve ethical problems" . An ethiDo we need the services of hospital ethics committees or consultants in African hospitals or at the bedside? That may not be the point presently. However, clinical ethics is neither about committees and consultations, nor about technology and end-of-life issues alone. Common sense and intuition are insufficient to address all ethical issues that arise in patients' care. The well-intentioned clinical decisions and judgments of yesterday may turn out to be unsound in the searchlight of today's ethical scrutiny. Although core moral virtues have generally guided medical practice in Africa as elsewhere, there is the increasing need to apply both cognitive and behavioural ethical values to everyday decision making at the bedside by the physician as well as other health-care professionals . PerhapsIt is not here suggested that next on the agenda of health care in Africa is to devote attention and resources to training bioethics consultants for the bedside. Most people in Africa still do not have access to qualified health personnel and reasonable health care. The point is that deliberate efforts should be made to train present and future health care providers to be aware of the core moral virtues required of them in their duties to patients; and be sensitive to the ethical values of their patients, their families and the society.In the developed world education in ethics is no longer a "hidden curriculum"  that is It is now time for Africa to join the rest of the world by introducing ethics education into the curricula of all medical schools where it is not presently taught. This is where the future of bioethics and health care delivery and research in Africa lies. Apart from some countries in the southern and eastern part of Africa and a handful of universities in other parts of Africa, there is no formal ethics education in most of Africa's medical schools. Ethics education to medical students is a necessary and required commitment to accomplishing an all-round training of the doctor whose decisions are both technical and moral. Much attention has wholly focused on the technical aspect of medical education, leaving the student to develop his or her moral attitudes passively through observation and intuition. Formal ethics teaching aims at equipping students with a common framework on which to reconcile patients' medical needs with their values, perceptions, situations and beliefs ; and mayPertinent to any discussion about teaching bioethics in Africa is the issue of shortage of trained bioethicists to fill the vacancies that would be created in academic institutions in many African countries. Apart from South Africa and a few others, most countries in Africa lack the requisite bioethics manpower that would be needed in the medical schools. Even in institutions where bioethics is already part of the medical curriculum, it is unlikely that there are enough bioethics teachers. It is in this regard that the efforts of international agencies that fund training of developing world bioethicists are noteworthy. Those Africans who have undergone bioethics training in the developed world and have become pioneers in their institutions have an awesome responsibility of establishing credible training agenda for their countries. They are also well positioned to directly seek funding for such home-based programmes from international sponsors. At the beginning, such scholars may encounter some crisis of identity and acceptance within the established academic system. However, such difficulties would fizzle out as they persist in highlighting and proffering solutions to the myriad of contemporary ethical problems within the system and the society. The initial difficulty of publishing their works in established western bioethics literature can be overcome by patronizing local or regional journals that target the primary audience for which their work is meant and open access journals that reach far and wide. Moreover, most bioethics literature consists of commentaries, observations, personal opinions and philosophical reasonings. As African boethicists embark on qualitative research to highlight ethical issues in Africa and provide quantitative data to fill the gap and provide information which are frequently lacking in most western bioethics journals, access to western dominated journals would be enhanced.Bioethics, Chadwick and Schuklenk question the altruism behind training developing world bioethicists in the west and warn against bioethics colonialism. They opine that graduates of such programmes are subjected to western ethical views and ideologies and that the developing world is not funded to develop bioethics capacity based on its own thinking [In an editorial published in a recent issue of thinking . While IIn the light of the foregoing, it is imperative that African bioethics must evolve which should take cognizance of its unique needs and circumstances and which, though amenable to improvement as a result of continuing interactions with other cultures and values, yet is not overshadowed by those influences. The need for the individual clinician/researcher to be committed to upholding high ethical standards and principles that respect the social, cultural, economic, educational and religious values of the people can not be over-emphasized. More efforts are required towards increasing continent-wide awareness about ethical issues in biomedical practice and research through ethics conferences, workshops, national bioethics conferences, the public media and Non-Governmental Organisations (NGOs). Countries where bioethics presence is fairly strong should assist neighboring countries to establish a presence, especially in organizing ethics review committees at research centres and institutions. Within countries, the possibility of joint or regional ethics review committees should be explored.Continuing and expanded support from the international bioethics community is required now more than before, to develop capacity for training of academic faculty, clinicians, researchers, government health ministry officials, NGOs, and the media in bioethics. The initiatives of the National Institute of Health of the United States to provide training grants for bioethics programmes within and outside Africa and the support of other institutions and bodies like the Wellcome Trust, African Malaria Network Trust (AMANET) and European Forum for Good Clinical Practice (EFGCP) among others, to the course of bioethics in Africa are noteworthy. Their support for bioethics capacity building programmes should not be limited to one or two sub-regions but to the whole continent. Further sponsorship should be provided for academic institutions within Africa to establish more short- and long-term training programmes at sub-regional levels. More importantly, these institutions should support the movement towards formal incorporation of bioethics into the curricula of medical schools and other health training institutions in Africa. The present and future needs for this in Africa are most apparent now. According to an African adage, the best time to plant a tree is twenty years ago, the next best time is now.The author was a Fogarty Fellow (2003/2004) at the Joint Centre for Bioethics, University of Toronto 88, College Street, Toronto, Ontario, Canada M5G 1L4.The pre-publication history for this paper can be accessed here:


Lack of antihypertensive use among hypertensive individuals is a major public health problem. It remains unclear as to how much of this lack of treatment is because of failure to diagnose hypertension or failure to initiate drug treatment for those with a diagnosis of hypertension. The primary aim of this study was to determine the proportion of those untreated individuals who would be recommended to start drug therapy for control of blood pressure among those aware or unaware of their diagnosis of hypertension.The Canadian Heart Health Surveys (1986 – 1992), a national, cross-sectional descriptive survey (n = 23 129), was used to determine the proportion of individuals who were untreated, yet satisfied the 2004 Canadian hypertension guidelines for initiating drug therapy. Patients were divided into subgroups of those aware and unaware of having a diagnosis of hypertension according to self reported awareness from the survey.Of those with untreated hypertension (= 140/90 mmHg), only 37% were aware of their diagnosis. 74% of untreated individuals aware of their diagnosis of hypertension would require drug therapy, compared to 57% of those who were unaware. Of those >65 years of age, 52% of aware individuals needed drug therapy whereas only 34% of unaware elderly would need drug treatment.In both unaware and aware subgroups, the majority of patients with untreated hypertension would benefit from antihypertensive drug therapy according to the 2004 Canadian Hypertension recommendations. The proportion of untreated patients that still need drug therapy was higher among those who were aware compared to those who were unaware. This finding suggests that the major gap in hypertension control may be in initiating drug therapy rather than in diagnosing hypertension. Further studies are needed to confirm these results to ultimately help strategize public health efforts in controlling hypertension. Antihypertensive drug therapy can reduce cardiovascular morbidity and mortality by 25–30% -3 in thoFrom the 2004 Canadian Hypertension Guidelines, pharmacotherapy is recommended if the diastolic blood pressure is > 100 mmHg; the systolic pressure is > 160 mmHg; or the diastolic blood pressure is >90 mmHg in the presence of cardiovascular heart disease or risk factors. To determine the proportions of individuals who satisfied the guidelines for initiation of drug treatment for hypertension, we used the Canadian Heart Health Surveys (1986 – 1992). This national, cross-sectional descriptive survey (n = 23 129) contains data on individual blood pressure measurements using standardized technique as well as information on cardiovascular risk profile, antihypertensive medication use and self reported awareness of hypertension. Further details of this survey have been published elsewhere [Of those with untreated hypertension (= 140/90 mmHg), only 37% (n = 897 786) were aware of their diagnosis. Among those aware and unaware of their diagnosis of hypertension, 35% vs. 36% were female; 14% vs. 12% had any cardiovascular disease and 31% vs. 75% were under the age of 65 years respectively. The proportions of those who would be recommended drug therapy among those who are aware and unaware are presented in Table In both unaware and aware subgroups, the majority of patients with untreated hypertension would benefit from antihypertensive drug therapy according to the 2004 Canadian Hypertension recommendations. This analysis extends the findings from other studies ,7,8 by dThere are several limitations with this study. First, the Canadian Heart Health Survey data are 12 years old and there is some evidence to suggest that prescription rates of antihypertensive drugs have improved since then . HoweverThis study found a considerable number of both unaware and aware patients with untreated hypertension require drug therapy. However, the majority of those who needed drug therapy were actually aware that they had a diagnosis of hypertension compared to those who were not aware. This finding suggests that the major gap in hypertension control may be in initiating drug therapy rather than in diagnosing hypertension. Further studies are needed to confirm these results to ultimately help strategize public health efforts in controlling hypertension.The author(s) declare that they have no competing interests.NK, DW and NC contributed to the design of the project. NK contributed to data analysis. NK, DW and NC contributed to writing the manuscript and for substantive, intellectual editing contributions.The pre-publication history for this paper can be accessed here: