It has been 50 years since chronic granulomatous disease was first reported as a disease which fatally affected the ability of children to survive infections. Various milestone discoveries from the insufficient ability of patients' leucocytes to destroy microbial particles to the underlying genetic predispositions through which the disease is inherited have had important consequences. Longterm antibiotic prophylaxis has helped to fight infections associated with chronic granulomatous disease while the steady progress in bone marrow transplantation and the prospect of gene therapy are hailed as long awaited permanent treatment options. This review unearths the important findings by scientists that have led to our current understanding of the disease. Landinget al.  also deset al. [.). This phenomenon was rapidly established as a sensitive clinical screening test for CGD that is still used today.In the 1960s, studies on patient blood confirmed CGD to be a disease of impaired phagocytes. Holmes et al.  suggesteet al. ,8. It wa. and other reactive oxygen species such as hydrogen peroxide (H2O2) [2O2 was illustrated by the fact that some bacterial species, such as Streptococci, that produces its own H2O2, could be killed by CGD leucocytes [., produced by NADPH oxidase during its respiratory burst, and showed that whereas normal leucocytes generated O2-. during phagocytosis, CGD phagocytes were not able to do this [Over the next twenty years, over 150 scientific papers had been published reporting cases of patients with CGD describing their symptoms and similarities to other granuloma forming diseases. It was observed that CGD leucocytes could ingest micro-organisms but failed to kill the organisms responsible for the infections. Phagocytosis is normally accompanied by a marked increase in oxidative metabolism and studies had shown that NADPH oxidase was the respiratory enzyme responsible for bactericidal activity . The hexe (H2O2) . The impucocytes . Followi do this -15.. generating activity began in 1979 from observations that a cytochrome-b associated with a particulate fraction of normal neutrophils, was absent from the neutrophils from some, but not all, patients with CGD [558 was proposed as a primary component of the microbicidal oxidase system of phagocytes. A multicenter European evaluation of its incidence and relevance was conducted in London [558 was hailed an important component of the microbicidal NADPH oxidase system and provided insight into its role in the enzyme complex. Borregaard and colleagues [558 resides in the membrane of the specific granules of unstimulated human neutrophils and that the cytochrome-b558 translocates to the plasma membrane when the cells are stimulated. The authors speculated that the observed translocation was essential to the formation of an electron transport chain which generates O2-., the single precursor from which all microbicidal oxidants ultimately arise.Speculation that a b-type cytochrome may also be involved in this O2-with CGD . Describn London  where itlleagues  demonstr558 was, so far, the only clearly defined component of this oxidase system and its absence provided the molecular basis of X-linked CGD, in which a profound predisposition to infection resulted from complete failure of this respiratory burst. Within a month of each other in March 1987, three separate groups working on the constituents of the phagocyte NADPH oxidase (phox) enzyme published their findings. Segal was the first to find that cytochrome-b558 had two subunits – a 23 K protein and the previously described 76–92 K glycoprotein. Reporting that the subunits were closely linked and remained associated with the heme of cytochrome-b558, neither protein was detected in the cells of five patients with X-linked CGD, whereas both were present in two with the autosomal recessive inheritance form of this disease. This was the first finding substantiating what we now know are the smaller and larger subunits of the phagocyte cytochrome-b558 heterodimer (p22phox and gp91phox respectively) [558 or not, and Curnutte and Scott [The 1980s saw the formation of a disease-gene relationship. Linkage analysis using cloned, polymorphic DNA probes suggested a proximal location Xp21) within CGD families [ctively) . Umei an within C558-positive X-linked form of CGD have been reported by others since [In 1981, a variant or atypical X-linked form of CGD was described  whereby rs since ,26. Todaet al. (1989) cloned and sequenced a cDNA encoding the 47-kD component of the NADPH oxidase system [et al. (1990) [et al. [558 and its chromosomal location, which served as a foundation for the analysis of genetic abnormalities at this locus in CGD. The cloning of p40phox by Wientjes et al. [In 1986, the gp91phox subunit was first cloned , encodede system , Leto et. (1990)  cloned a [et al.  reporteds et al.  provideds et al. .Research into CGD has improved our knowledge of the normal NADPH oxidase system. In some cases, the identification of specific mutations has provided insights at a molecular level. Most patients with CGD have mutations in the CYBB gene that encodes gp91phox, located at Xp21.1. Since the discovery of the CYBB gene no single mutation appeared to be responsible and a flood of research on mutations ensued, reporting large and small deletions, frameshifts and other mutations. The genetics of many hundreds of families with CGD were investigated and recounted and a mutation registry database for X-linked and autosomal recessive CGD was set up by Dirk Roos and MaunoVihinen from the University of Tampere in Finland  which prAutosomal recessive CGD arises from mutations occurring in either the p67phox, p47phox or p21phox phagocyte oxidase proteins. Mutations in the NCF2 gene which encodes the p67phox represents about 5% of CGD cases whereas deficiencies of NCF1 which encodes p47phox represents about a quarter of all cases of CGD . Unlike . generating complex is confined to the plasma membrane [The dormant oxidase consists of both cytosolic and membrane-bound components, but the active O2-membrane ,2. This 558 is present but does not transfer electrons, demonstrating that the absence of translocation in other forms of CGD is not a secondary effect of the failure to generate O2-. [The 47 kDa phosphoprotein was found in both the cytosol and membranes after stimulation of neutrophils with phorbol myristate acetate and the phosphoprotein was detected in the cytosol preceding its detection in membranes . Activatate O2-. . Ding anate O2-.  were unaate O2-. . A schem., which subsequently is dismutated into H2O2., In the presence of myloperoxidase which is delivered by primary granules into the phagosomes, H2O2 gives rise to more potent oxidants such as oxyhalides (most frequently hypochlorous acid), the hydroxyl radical (OH-.), or singlet oxygen (O-.) [The primary biochemical defect in CGD that leads to impaired microbicidal activity was thought to be failure of phagocytes to generate sufficient quantities of reactive oxygen species (ROS), which are responsible for the so-called respiratory or oxidative burst. These compounds are derived from initial production of the extremely unstable and weakly bactericidal O2-en (O-.) . The hig+-rich and alkaline environment in the phagocytic vacuole. Thus, the role of ROS in the killing process is not as direct as previously thought, although, ROS are necessary to increase phagosomal osmolarity. Mice made deficient in neutrophil-granule proteases, but normal in respect of superoxide production and iodinating capacity, were unable to resist staphylococcal and candidal infections, suggesting that proteases are primarily responsible for the destruction of the bacteria [Ca channel was blocked, revealing an essential and unexpected function for this K+ channel in the microbicidal process [The fact that phagocytes kill microbes through toxic oxygen radicals and their metabolites provided much of the biological basis for theories relating the toxicity of oxygen radicals to the pathogenesis of CGD. However, recent evidence  indicatebacteria . Further process .In addition to recurrent life-threatening infections, patients often develop sterile granulomas in hollow organs, liver, lymphoid tissue and skin, without clinical evidence of infections . The mec. production and Staphylococcus aureus bacterial killing. In two patients with variant X-linked CGD, rIFN-γ treatment in vivo was also associated with increased spectral levels of neutrophil cytochrome-b558 [. production following rIFN-γ therapy, where O2-. production was not sustained or associated with any change in cytochrome-b558 levels [et al. [558 gene expression in Brazil [In the late 1980s the potential of interferon gamma was investigated as a prophylactic therapeutic agent for CGD as preliminary studies in CGD patients demonstrated that brief in vitro or in vivo administration of recombinant IFN-γ (rIFN-γ) significantly enhanced phagocyte O2-ome-b558 . Subsequ8 levels . In some [et al.  showed t [et al. . Recent n Brazil  found inFollowing the successful use of allogeneic bone marrow transplants to treat children with severe combined immune deficiency . GoudemaAspergillus fumigatus, Burkholderia cepacia and Staphylococcus aureus [+ cells obtained from X-CGD patients. It was shown that the level of O2-. production in phagocytes derived from transduced cells was 69% of normal levels [Gene therapy involves the permanent genetic correction of hematopoietic stem cells in which a vector is used to carry the corrective gene into the cells which are then re-introduced to the body. CGD is a particularly good candidate for gene therapy because low levels of functional phagocytes are expected to provide significant activity against pathogenic microbes. In 1992, Thrasher and colleagues  used cels aureus . These sl levels . In the l levels  isolatedl levels . This suGene therapy trials for CGD in Europe began in 2004 through the collaborative efforts in Frankfurt and London. So far, two patients have been treated with retroviral vector and encouraging results show that both patients have significant levels of gene corrected cells and are clinically well . Gene therapy holds great promise as an alternative treatment for patients without suitable marrow donors or where BMT is not a viable treatment option.It is estimated that one in 250,000 babies is born with CGD, although symptoms of the condition may not appear until after the first few months of age. Early reports documented the survival of patients beyond 7 years of age as 21%  which haThe author(s) declare that they have no competing interests.


With the prospect of another pandemic Influenza fresh in our consciousness, the pathogenic nature of the Influenza A virus and its ability to induce high rates of mortality are ever more pertinent. Recently a novel protein encoded by an alternate reading frame in the PB1 Gene segment of Influenza A virus has been discovered and in turn shown to enhance viral virulence in a mouse model . This re An intrinsic property of all viruses is their need to either subvert and/or subdue the host immune response to establish a productive replication cycle. The mechanism by which this avoidance proceeds is greatly varied throughout the phyla of viruses; however either must occur in order for a virus to spread and propagate. The enhancement of this ability to subvert and disable host immunity is directly correlated to increased viral pathogenicity in the host . For exaOther additional mechanisms of host defense manipulation and avoidance by Influenza A virus exist including a novel alternate reading frame recently discovered in the PB1 polymerase gene segment. This reading frame is found in select Influenza A viruses and has been shown to impact host defense mechanisms and in turn enhance pathogenicity in vivo . It has The discovery of a novel protein that contributes to an increase in Influenza A virus pathogenicity is of great concern due to the impact that Influenza A virus has on both human health as well as on the economy. Globally it is estimated that between 300,000 to 500,000 people may die annually due to influenza virus infections . In the Specifically, Influenza A virus is an enveloped, eight segmented minus-sense RNA virus, that is a member of the orthomyxoviridae genus of viruses. Unique to Influenza A virus, compared to other lytic mammalian RNA viruses, is that all mRNA synthesis and genome replication occurs in the nucleus of the infected host cell . FurtherStreptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae [Despite Influenza A virus's speedy replication cycle and cytolytic properties, the major cause of death associated with an Influenza A virus infection is secondary bacterial pneumonia. The major pathogens associated with this complication are fluenzae . One of fluenzae . The mucfluenzae . However+ TH cells. This antigen presentation results in clonal expansion of a given CD4+ TH cell lineage and subsequently a range of downstream effector functions are induced, such as cytotoxic T lymphocyte (CTL) activation, antibody production, and inflammation [Besides the destruction of mucus producing lung epithelia cells, another possible contribution to secondary bacterial infection as the result of Influenza A infection is the targeted elimination of alveolar macrophages by the Influenza PB1-F2 protein. Initially precursors to macrophages, monoblasts, are produced in the bone marrow and secreted into the bloodstream where they become monocytes. Once monocytes exit the circulation and migrate into tissue, they differentiate into tissue specific resident macrophages, such as Kuppfer cells in the liver or alveolar macrophages in the lung . These rammation . By PB1-+ CTL on the surface of infected cells. After scanning the Influenza A genome for this novel antigenic, CTL-activating peptide, it was found to correspond to a protein encoded by an alternate reading frame found within the PB1 gene segment [The initial discovery of the PB1-F2 protein by Chen et al. occurred via a broad search for antigenic Influenza A viral peptides that are encoded by alternate reading frames. Chen et al. also examined if these novel polypeptides are presented by major histocompatibility complex I and in turn recognized by CD8 segment . Ribosom segment . When an segment . When lo segment . PB1-F2  segment . Interes segment . It has in vitro findings strongly suggested that the presence of the PB1-F2 protein would have an increasing effect on viral pathogenicity in vivo. This hypothesis was confirmed by a recent study conducted by Zamarin et al., which examined PB1-F2's contribution to Influenza A virus pathogenesis in a mouse model. Indeed a virus expressing the PB1-F2 was found to have an increased virulence when compared to a knockout virus lacking the alternate reading frame. Also the presence of higher viral titers in the lungs of mice infected with a PB1-F2 expressing strain demonstrated a reduction in the animal's ability to clear the virus as well as mount an effective immune response [In sum, these Interestingly this alternate reading frame is found in the pandemic strains of 1968, 1957, and the infamous 1918 strain [Currently it has been found after large-scale, exhaustive sequence analysis of avian Influenza A virus isolates that the PB1-F2 transcript is under the highest positive selective pressure for nonsynonymous substitutions . This co


Daily counts of preterm births were obtained from birth records in four Pennsylvania counties from 1997 through 2001. We observed increased risk for preterm delivery with exposure to average PM10 and SO2 in the 6 weeks before birth , adjusting for long-term preterm delivery trends, co-pollutants, and offsetting by the number of gestations at risk. We also examined lags up to 7 days before the birth and found an acute effect of exposure to PM10 2 days and 5 days before birth  and SO2 3 days before birth , adjusting for covariates, including temperature, dew point temperature, and day of the week. The results from this time-series analysis, which provides evidence of an increase in preterm birth risk with exposure to PM10 and SO2, are consistent with prior investigations of spatial contrasts.Preterm delivery can lead to serious infant health outcomes, including death and lifelong disability. Small increases in preterm delivery risk in relation to spatial gradients of air pollution have been reported, but previous studies may have controlled inadequately for individual factors. Using a time-series analysis, which eliminates potential confounding by individual risk factors that do not change over short periods of time, we investigated the effect of ambient outdoor particulate matter with diameter ≤10 μm (PM Preterm delivery can lead to neonatal mortality as well as an array of infant morbidities that range from pulmonary to neurologic outcomes . The pre10), and in some cases total suspended particulates (TSP)—and preterm delivery by examining spatial exposure contrasts between individuals based on the place of residence at birth . These counties were chosen for their diverse mix and wide range of pollutants. Births were restricted to gestational ages within the range of 20–44 weeks because births at < 20 gestational weeks are rarely viable and births are usually induced before 44 weeks of gestation.The study population consisted of all live singleton births whose mothers resided in four Pennsylvania counties  and for whom birth certificates were filed with dates of birth between 1 January 1997 and 31 December 2001 (10 and SO2) and for those considered co-pollutants . PM10 was collected hourly for three of the counties using a tapered element oscillating microbalance sampler, and every sixth day in one county using a high-volume air sampler system. We used public access data from EPA’s AQS and did not directly measure pollutants with these samplers. Thus we do not know the manufacturer for the TEOM and high-volume air samplers. Three counties had multiple monitoring stations for each pollutant; a single average countywide daily pollutant value was computed for each of these counties.We obtained ambient outdoor air pollution monitoring data for the four study counties over the 5-year study period (20 November 1997 through 30 December 2001) from the U.S. Environmental Protection Agency (EPA) Air Quality System (AQS). Daily mean values were computed for the two air pollutants of primary interest , which also change over short periods of time. These meteorologic data were obtained from the National Weather Service .Gestational age was computed as the number of weeks between the date of the last menstrual period (LMP) and the date of birth. For birth records missing the date of the LMP, the clinical estimate of gestation was used. Eligible births with gestational ages < 36 weeks were considered preterm. Counts of preterm births were tallied for each day during the 5-year observation period.10 and SO2 levels and counts of preterm births jointly over time. Preterm births were measured as a daily count of events assumed to be independent and random. The Poisson model was selected because these counts were small on any given day. Because days of observation were the units of analysis, exposure gradients were measured with reference to time rather than to other individuals.We conducted a Poisson regression analysis, which followed PMa priori because of their established temporal relationship with air pollution in the previous literature and their likely associations with the air pollutants of interest. Previous studies also suggest a consistent seasonal pattern for preterm birth , were included rm birth .2, and SO2 in the PM10 analysis and CO, NO2, and PM10 in the SO2 analysis). Because the number of births in the population varies by season, we included a term in the time-series model to offset the total number of gestations in the population at risk for preterm birth on each day during the study period.We used daily pollutant levels in each county to compute the mean air pollution concentration value for the 6-week period preceding each day of observation. To incorporate county-level information, we used a multivariable mixed-effects model with a random intercept for each county. We controlled for long-term preterm birth trends and mean 6-week level of co-pollutants  and 95% confidence intervals (CIs) for 6-week and daily acute exposure (for each of the 7 days before birth) to PMApproval for this research was obtained from the Committee on the Protection of the Rights of Human Subjects at the Biomedical Institutional Review Board of the University of North Carolina at Chapel Hill’s School of Medicine.A total of 200,253 birth records were available for the four counties between 1997 and 2001. For 36,839 birth records missing the date of the LMP, we used the clinical estimate of length of gestation; secondary analyses showed that inclusion of these births did not meaningfully change the estimated associations but did enhance precision, and we therefore included these births in the final analyses. We excluded 677 births to mothers whose geocoded residence at delivery was outside the four study counties and 6,322 multiple births. An additional 5,257 births with gestational ages outside the range of 20–44 weeks were excluded. After these exclusions, 187,997 births  remained. 10 and SO2 exposure and risk for preterm delivery. Risk for preterm birth increased with exposure to mean 6-week PM10 , offsetting by the total number of gestations at risk, and adjusting for county-specific long-term trends and co-pollutants . The RRs for the two higher PM10 exposure quarters were slightly elevated, but there was no clear evidence of a strictly monotonic dose–response relationship between 6-week PM10 exposure and preterm delivery in the quartile analysis.10 levels using a 2-day lag  and a 5-day lag   .2, the lag with the strongest association was 3 days  (For SO 15 ppb) .2 and PM10. Our point estimates are similar to those reported in the literature, and the widths of our CIs compare favorably with those for previously reported estimates. The estimated number of excess preterm births that can be attributed to these pollutants, computed as the number needed to treat (NNT)  = 0.114] and given a RR = 1.07 for PM10, the risk among exposed (R1) = 0.122, the RD (risk difference) = 0.008, and the NNT = 1/0.008 = 125. In the week before birth, the strongest associations were observed with a 2-day and 5-day lag for PM10 and a 3-day lag for SO2.We observed an increased risk for preterm delivery during the last 6 weeks of pregnancy with exposure to SOat (NNT) , was app2 have been observed   = 1.10; 95% CI, 1.01–1.20] for each 100-μg/m3 increase in TSP  for each 100-μg/m3 (37.5 ppb) increase in SO2. A retrospective study conducted among 126,752 singleton births in the Czech Republic during 1991 reported adjusted ORs for preterm delivery to be 1.12  and 1.24  per 50-μg/m3 (18.75 ppb) increase in TSP and SO2, respectively, during the third trimester of pregnancy  during the last month of pregnancy (Our results are consistent with a number of prior studies of PMin PM10) . This ste in TSP . This stregnancy . In Vancregnancy .A potential limitation of these previous studies is inadequate control for confounding by individual risk factors. Birth record data do not include information on all risk factors that could potentially confound the relationship between air pollution and preterm delivery. In addition, variables that are collected may be poorly measured or recorded, particularly when they come from a birth certificate, which could lead to residual confounding in either direction of the association between air pollution and preterm birth.By observing the population over time using time-series analysis, we could investigate the impact of air pollutants without the influence of known and unknown individual risk factors that do not vary over short periods of time. Consistency of the overall findings of our study with previous studies suggests that confounding at the individual level is probably not explaining the observed association between air pollution and preterm delivery. However, more complete control of confounding could explain the weaker associations observed in this study; this could also be caused by differences in pollution levels or sources between study locations.Our study was efficient in that it made use of existing air pollution and birth record data. This allowed for a very large, population-based study that is not subject to selection biases that arise when recruiting a patient population. In addition, the long study period and large population enhanced the precision of the estimated measures of effect. Given these advantages, in addition to the ease associated with using extant data sources relative to generating original data, investigation of additional health outcomes (including other birth outcomes) using this study design is advisable.An acknowledged limitation of all studies of ambient air pollution is that measurements from stationary outdoor monitors may not represent individual exposure. Studies of air pollution and mortality, cardiovascular disease, and other respiratory outcomes have addressed this issue and report that, although relatively crude, ambient measures are often the most feasible measure of exposure in terms of cost and burden to the study participant . In addiThe time-series analysis assumes that ambient pollution levels and personal exposure are often correlated over time, so although their absolute levels may be different, both will be high on a high-air-pollution day and low on a low-air-pollution day . Measure10 and SO2 are hazardous themselves or are markers for other hazardous airborne pollutants, such as sulfates that result from SO2 emissions in the presence of water and oxygen, fine particles (PM2.5), or other noncriteria pollutants such as polycyclic aromatic hydrocarbons, was beyond the scope of this study and is a direction for further investigation. Some of the previous literature suggests associations between CO and/or NO2 and preterm birth . The question of whether PMrm birth . We focu10 limited the number of observation days for both the 6-week and daily acute effects analysis, reducing the precision of the effect estimates.Missing data for PM10/TSP at beginning of pregnancy (first month and first trimester) and preterm delivery  during pregnancy for the effect of air pollution on preterm birth, and to delineate the biologic mechanism for such an effect would also be useful.Despite limitations in our data, we observed evidence of an effect of both PM


UL97, however additional mutations that also confer resistance to the drug were mapped to UL27. These open reading frames share a low level of homology, yet the function of pUL27 remains unknown. A recombinant virus with a deletion in the UL27 open reading frame was reported previously to exhibit a slight replication deficit, but a more important function in vivo was hypothesized given its homology to the UL97 kinase. The potential for an important function in vivo was investigated by determining if these knockout viruses could replicate in human tissue implanted in SCID mice. None of the AD169 derived viruses replicated well in the implanted thymus/liver tissue, and is consistent with previous observations, although all of the viruses replicated to some degree in retinal tissue implants. Replication of the parent viruses was observed at 7 days post inoculation, whereas no replication was detected with any of the recombinant viruses with deletions in UL27. By day 14, replication was detected in two of the three knockout viruses and in all of the viruses by day 42. These data are consistent with minimal defects observed in cell culture, but are not consistent with an important role for UL27 in vivo. We conclude that UL27 is not required for viral replication in vivo.Inhibition of the human cytomegalovirus UL97 kinase by maribavir is thought to be responsible for the antiviral activity of this compound. Some mutations that confer resistance to maribavir map to  UL97. A recombinant virus that does not express this kinase [UL27 was reported to be a paralog of UL97 and shares 12% identity at the amino acid level [UL27 replicate as well as the parent virus, but recombinant viruses containing large deletions in the open reading frame exhibit a slight, but repeatable growth defect of about half a log in single step growth curves [UL27 might be more apparent in vivo and sought to characterize its replication in established models of infection using implanted human tissue in SCID mice.Although human cytomegalovirus (HCMV) infections are common in the general population, they can cause significant morbidity and mortality in immunocompromised individuals . Severals kinase  has beens kinase . A recoms kinase . Recentls kinase ,5. This UL27 were examined. The construction and growth characteristics of T2092-1-1-7 were described previously [UL27 and the carboxyl terminal portion of UL28 (coordinates 32884–34674) including the entire region deleted in T2092-1-1-7. A second virus, 1/3ΔUL27BAC, contains a mutation that that deletes the central third of UL27 without disrupting UL28 (coordinates 33452–34053). To confirm the genomic structure of the engineered genomes, restriction fragments were compared to the AD169-BAC and the predicted changes in fragment sizes were observed. No other alternations in the BAC DNA were apparent. The coordinates of the deletions were also confirmed by PCR and sequencing of the PCR products flanking the deletion region. Both of these viruses were similar to T2092-1-1-7 in that they both exhibited modestly reduced replication in cell culture and attained titers approximately 10-fold lower than the control virus. Since the same phenotype of reduced replication efficiency was observed in all three the recombinant viruses, we conclude that it is likely related to the engineered mutations in UL27.Three viruses with deletions in Each of these viruses and the parent viruses from which they were derived, were used to inoculate SCID mice containing implanted retinal tissue or thymus/liver tissue . Briefly10PFU/g by 42 days post inoculation  declare that they have no competing interests.MNP provided critical intellectual input and analysis of the studies. DQ provided critical intellectual input and conducted some of the animal studies. DJB conducted in the animal experiments SC, GK and JCD provided recombinant viruses and provided critical intellectual input. ERK provided critical intellectual input.


Cell lines have been used to study cancer for decades, but truly quantitative assessment of their performance as models is often lacking. We used gene expression profiling to quantitatively assess the gene expression of nine cell line models of cervical cancer.We find a wide variation in the extent to which different cell culture models mimic late-stage invasive cervical cancer biopsies. The lowest agreement was from monolayer HeLa cells, a common cervical cancer model; the highest agreement was from primary epithelial cells, C4-I, and C4-II cell lines. In addition, HeLa and SiHa cell lines cultured in an organotypic environment increased their correlation to cervical cancer significantly. We also find wide variation in agreement when we considered how well individual biological pathways model cervical cancer. Cell lines with an anti-correlation to cervical cancer were also identified and should be avoided.Using gene expression profiling and quantitative analysis, we have characterized nine cell lines with respect to how well they serve as models of cervical cancer. Applying this method to individual pathways, we identified the appropriateness of particular cell lines for studying specific pathways in cervical cancer. This study will allow researchers to choose a cell line with the highest correlation to cervical cancer at a pathway level. This method is applicable to other cancers and could be used to identify the appropriate cell line and growth condition to employ when studying other cancers. Howevare lost ,4. Therein vitro research of cervical cancer involves the culture of immortal cervical cell lines in monolayer [in vivo phenotypic structure of epithelial tissue and has been used in different types of research, including breast [Current onolayer ,5. Cell g breast ,7, skin g breast ,9, cervig breast , and heag breast  cancer, g breast  and indig breast ,13.et al. used the NCI 60 microarray data to compare cell lines in culture to their respective cancer microarray data sets. They performed a singular value decomposition (SVD) cluster analysis and generated a quantitative value termed the tissue similarity index (TSI), which denotes how well various cell lines still represent their tissue of origin [Few attempts have been made to quantitatively assess how closely cell lines actually model tissue. Most results from analyses such as clustering of expression profiles of cell lines and tissues simply conclude that cell lines resemble their tissue of origin . For exaf origin .With this in mind, we wanted to quantitatively assess how well cervical cancer cell lines commonly used to study cervical cancer actually model the disease, focusing on late-stage invasive cervical cancer. Therefore, the gene expression profiles of nine different cervical cell lines were correlated separately to the expression profiles of nine cervical cancer and three normal cervical biopsies. In addition to their American Type Culture Collection (ATCC) recommended culture media, HeLa and SiHa cell lines were additionally cultured in different media as well as in an organotypic environment to assess how their correlation to tissue changes in different culture environments. Conditions with higher correlations indicate better models of cervical tissue. In order to move beyond a simple global comparison, the correlations of each condition were also compared to cervical tissue at individual pathway-specific levels. This information provides a more detailed view of our ability to model cancer using cell lines.Before comparing cervical cancer transcriptional profiles with cell line profiles, we first determined that the biopsies used in the analysis accurately represented cervical tissue and invasive cervical cancer. We tested the differentially expressed genes between normal and cancerous biopsies by first examining transcriptional changes in light of presumed biological mechanisms of cancer biology; we then compared the differentially expressed genes with regard to previous cervical cancer research.Benjamini-Hochberg multiple hypothesis correction (p < 0.01) and a 2-fold minimum expression change was used to identify 140 genes that were differentially expressed between cervical cancer and normal cervix. The 140 genes [see Additional file We first used presumed biological mechanisms of cancer biology to assess our differentially expressed genes. First, groups of genes that represented common cancer pathways were checked for correct expression changes ,17. A t-A second analysis provided further evidence that the expression profiles in these cervical cancer biopsies were consistent with previous observations in the literature and therefore suitable for further detailed analysis. Approximately 650 differentially expressed genes identified in the literature, derived from small scale microarray studies -22, diffA transcriptional comparison of cervical tissue to cell lines was performed using hierarchical clustering Figure  and SVD We recognize that the particular cervical cancer biopsy samples that we used in this study do not represent the diversity of all possible cervical cancer samples. However, the comparisons described above provide strong evidence that the expression profiles collected accurately represent both cervical cancer and normal cervix. Once the reliability of the expression profiles was established, they could be used to compare the cell line expression profiles to identify which of the tested cell lines is a better model of cervical tissue.The correlation of cell lines to both cervical cancer and normal cervix were calculated to evaluate how well different cell lines and culture conditions modeled the gene expression programs in cervical cancer. The global correlation of transcriptional profiles provided a quantitative assessment of how well cell lines model tissue; a higher Pearson correlation coefficient denoted a better model of cervical tissue. The correlations for each cell line and growth condition were summarized in Figure in vivo environment. HeLa cultured as a monolayer had a surprisingly poor correlation to cervical cancer, given that it has been extensively used in cancer research [in vivo environment. It is possible that culturing the primary, C4-I, and C4-II cell lines in an organotypic environment would further increase their correlation to both cervical cancer and normal cervix.The correlation of normal to normal tissue or cancer to cancer tissue samples was calculated to provide a best-case scenario for the cell line correlations and to provide an estimate of patient variation. These results also provided a measure of how well cell lines can be expected to model tissue in general. The Pearson correlation coefficient among cervical cancer specimens was 0.81 while the correlation among normal cervix specimens was 0.83, setting the upper expected limit of this measure. The correlation of cervical cancer to normal cervix was higher than any cell line at 0.62. Of the cell lines we tested, the primary cell line was found to be a better model than others, of both cervical cancer and normal cervix in terms of overall mRNA expression correlation. The primary cell line was expected to have the highest correlation to cervical cancer over the other cell lines because it was more recently out of its research -34. HeLaOnce we had identified a cell line with the highest correlation to cervical cancer (C4-I), we generated a list of 196 differentially expressed genes between the C4-I and primary normal cell lines. This list was used to hierarchically cluster the normal cervix and cervical cancer biopsies. The resulting dendrogram [see Additional file This quantitative analysis provided information on how changes to the culture environment can change a cell line's performance as a model to cervical cancer. To provide a deeper analysis of how well cell lines model tissue, the correlation of each Gene Ontology (GO) pathway was calculated to both cervical cancer and normal cervix for each cell line and growth condition.BAG1, BFAR, BIRC1, BIRC3, BIRC4, MALT1, PRKCZ, and TNFAIP3, and had a 2–7 fold difference when compared to cervical cancer. This pathway failed to achieve significance when calculating individual pathway correlations, but was strong enough to lower the correlation for C4-II in the "Regulation of Apoptosis" pathway calculation. Interestingly, the HeLa cell line cultured in an organotypic environment had a correlation of 0.6 for the "Regulation of Apoptosis" pathway, whereas its global correlation to cervical cancer was 0.42. This pathway was not an exception, the organotypic HeLa cell line had a stronger correlation (R = 0.59) for six apoptotic pathways than even the primary cell line (R = 0.56).The global correlation of cell line models to tissue provided an analytical way to choose better overall models of cervical tissue; however, choosing an appropriate model for a specific pathway may be of more utility. Using GO annotations, the correlation for each pathway and for all cell lines and growth conditions was calculated against both cervical cancer and normal cervix. As shown in Figure GNG11, CXCL1, FZD2, GNA12, CALU, GPR19, AKAP12, GRINA, 2 ESTs, CALM2, GNAI2, DGKD, and EDNRB, and had a 3–50 fold difference when compared to cervical cancer.Of even greater importance, some cell lines had negative pathway-specific correlations to cervical cancer. For example, HeLa cells cultured in monolayer had a negative (-0.3) correlation to cervical cancer in the pathway "G-Protein Signaling" (GO:7186) Figure . Fourteein vitro. The example where HeLa had a negative correlation to the "G-Protein Signaling" pathway is extremely important, indicating that this system is dysregulated in these cells relative to cervical cancer. Research on this pathway involving HeLa cells as a model may draw inconclusive results. Care must be taken to identify which cell line and growth condition would yield the most appropriate model. The results here can not be quantitatively represented by cluster analyses such as SVD. Therefore, the pathway analysis was of great use when determining which cell line should be used to model cervical cancer or normal cervix at a pathway-specific level.Researchers studying a particular gene or pathway may not be interested in the best global model if that cell line does not represent their pathway of interest. Only cell lines with the highest correlation should be used to study a specific pathway In order to identify pathways that require more attention when selecting a cell line model, the correlations of all cell lines to cervical cancer for a specific pathway were averaged to generate a single correlation that represented all cell lines and growth conditions Figure . BiologiA pathway analysis of normal cervix versus cervical cancer is shown in Figure in vivo environment for decades and as a consequence have adapted to their new environment, resulting in changes of gene expression. We expected to see many important pathways with low correlations to tissue, and this was the case. We observed poor correlation to the cell cycle, RNA processing, and cell signaling pathways in cell lines compared to cervical cancer. This was due perhaps to both accumulated mutations and the fact that cultured cell lines may have different modes of cell-to-cell communication. These pathways, with low correlation to tissue , are extensively studied in cancer research [Most of these cell lines have been outside the research -37. TherPDK4. Pyruvate dehydrogenase kinase has been found to increase its expression during starvation [PDK4 was found to have a 4-fold decrease in expression in cervical cancer, a 2-fold increase in expression in HeLa cultured with DMEM, and a 20-fold increase in expression in HeLa cultured with MEM. As the function of PDK4 is the regulation of glucose metabolism, this indicates HeLa cultured in MEM experience starvation-like conditions, whereas HeLa cultured in DMEM have an environment similar to in vivo cancer conditions. This example of PDK4, along with the three metabolic pathways shown in Figure HeLa and SiHa cell lines were cultured in a different type of media to assess whether small changes to the environment could have a large impact on the correlation to tissue. The effect of different culture media on the correlation to cervical tissue was assessed at the pathway level using both SiHa and HeLa cell lines. An example of three biologically relevant pathways that changed in correlation between the two media is shown in Figure arvation . In our Organotypic culture physically resembles the structure of cervical epithelium. HeLa and SiHa cell lines increased the correlation to tissue when cells were cultured in organotypic cultures versus monolayer. The organotypic control, which consists of the same culture environment minus the fibroblasts, also increased the correlation over the cell lines, indicating a simple collagen bed was sufficient to increase the global modeling of cell lines to cervical cancer. In the case of the organotypic and organotypic control, the two environments did not differ significantly in their correlations Figure .Further evidence that organotypic cultures were better models over monolayer culture was provided by calculating the overall shift in pathway correlations for different environments, plotted as a histogram in Figure CYR61 had a dramatic decrease (7 fold) in expression. There was an increase in expression in many genes whose function was integral to the plasma membrane, including SLC7A11, SLC04A1, CLDND1, IER3, HOMER1, and AOC3. Many of these genes play a role in metabolic signaling or plasma membrane transport and were possibly up-regulated due to the increased communication between cells in this 3-dimensional culture. Many of the gene changes that allowed a cell line to grow in 3-dimensions were involved in cell-cell signaling pathways  was analyzed as to whether 3-dimensional culture conditions can improve the modeling performance of cell lines Figure . The "CeExpression profiles of cervical cancer biopsies were compared to previous cervical cancer research to provide evidence that the expression profiles accurately represent both cervical cancer and normal cervix. Primary normal cells and the C4-I and C4-II cells lines were found to be better models than the other cell lines we tested, even the more commonly used HeLa cell line. We found that simple changes to the environment, such as media, increased the correlation of HeLa and SiHa cells to cervical cancer. In addition, culturing HeLa and SiHa cell lines in an organotypic environment rather than in monolayer significantly increased their correlation to cervical cancer. The correlation of each cell line and growth condition was also analyzed at the pathway level. Despite the fact that many cell lines still retain a high expression correlation to cervical cancer, our pathway level analysis also revealed cell lines that had an anti-correlation to cervical cancer. Cell lines with low correlations to cervical cancer should be avoided in future studies as models of this disease.Nine cell lines were cultured in monolayer as well as under various perturbations, such as different media and structural environments. All cell lines except the primary normal epithelial line were obtained from the ATCC and cultured in ATCC recommended media. In addition to the recommended media, HeLa and SiHa cells were also grown in DMEM to assess how media changes their correlation to cervical cancer. Cell lines were cultured in 10% fetal bovine serum (FBS) (ATCC) and 1:100 PenStrep (Invitrogen). The primary cell line was a gift from Dr. Rebecca Richards-Kortum's lab at Rice University, and was grown in basal media and growth factors . All cell lines were cultured three times independently across three separate passages except the primary line, which was cultured at the same time in three separate plates due to the cells' short life spans. The cell line media conditions are summarized in Additional file 5/ml in 3 ml of collagen. 120 μl of the fibroblast/collagen suspension was added to each transwell plate (3 μm pore size and 6.5 mm diameter) (Corning) and incubated for 30 minutes at 37°C to solidify the collagen. SiHa and HeLa cells were resuspended at a concentration of 1 × 106/ml in DMEM media and 80 μl were added on top of the solid fibroblast/collagen suspension. 600 μl of DMEM media was added to the outside of the transwell insert and was replaced every other day. The organotypic control cultures were treated identically to the organotypic cultures, but without adding fibroblasts to the collagen; the absence of fibroblasts prevented 3-dimensional growth of the epithelial cell line.Organotypic cultures consisted of NIH 3T3 fibroblasts, collagen, and an epithelial cell line. Collagen was prepared by adding 2.2 ml of type I rat tail collagen (3 mg/ml) (Roche), 220 μl of 10 × DMEM, 220 μl of FBS, and Hepes-NaOH for a final pH of 7.2. Fibroblasts were resuspended in prepared collagen at a cell density of 3 × 10Three normal and nine moderately to poorly differentiated, invasive cervical cancer biopsies were obtained from the Cooperative Human Tissue Network (CHTN) and the Gynecologic Oncology Group (GOG) with the approval of the IRB at The University of Texas at Austin. Two technical replicates from each patient biopsy were hybridized separately to microarrays.Total RNA was isolated using Trizol (Invitrogen). RNA quality and quantity was assessed by gel electrophoresis and UV spectroscopy. DNA contamination was removed by RNeasy MinElute Cleanup (Qiagen). Universal Human Reference (UHR) RNA (Stratagene) was used as the reference channel in all hybridizations. RNA was amplified using T7 Message Amp (Ambion).Microarrays were printed on poly-L lysine coated slides with 47,000 previously sequence-verified IMAGE clones (Research Genetics/Invitrogen) on each slide, as described by Gu and Iyer . The ref2-transformation and background subtraction, data were normalized to a median intensity ratio of one. Only 8,338 genes with expression measurements on at least 80% of arrays were analyzed further. Averaged linked clustering and data centering was performed with LAD.Microarray images were processed using GenePix 4.0 software (Axon). After aligning the settings file and collecting the pixel intensity, the data were uploaded to the Longhorn Array Database (LAD)  for spotCalculation of the Pearson correlation between cell lines and tissue included the same data filtering described above; in addition, genes were also excluded if they were not present in at least 2 out of 3 cell line replicates. Replicates were averaged before calculating the correlation.Two separate SVD analyses were performed, first on all samples and second solely on the cervical tissue to identify genes differentially expressed between normal cervix and cervical cancer. In both cases, SVD was performed on the 8,338 genes used for hierarchical clustering where the non-missing row average for a gene replaced any missing data for that gene. The columns (cell lines or tissue biopsies) were normalized. 499 genes were identified as differentially expressed between cervical cancer and normal cervix by rank ordering the genes according to fold change, and selecting genes with a greater than 2-fold change.Benjamini-Hochberg multiple hypothesis correction (p < 0.01) with a minimum 2-fold expression change. The overlap of the SVD and t-test data produced a highly confident gene list of 140 genes. Additionally, a t-test was also used to identify 77 differentially expressed genes between SiHa cells grown as a monolayer versus an organotypic environment (p < 0.001).A Student's t-test was performed on the 8,338 genes described above to identify differentially expressed genes between cervical cancer and normal cervix. 434 genes were identified using a The Pearson correlation coefficient was calculated between each cell line and, individually, against cervical cancer and normal cervix. Genes were subjected to the same filters used in the SVD analysis. Tissue to tissue comparisons were calculated by averaging each replicate and then splitting the patient samples into two groups.Bonferroni multiple hypothesis correction was used  based on the database size of GO.The same data used to calculate the global correlation were used to calculate the pathway specific correlation. Replicates were averaged and the clone identifiers were mapped to LocusLink identifiers using SOURCE . Clone IMWC carried out the cell culture, tissue procurement, mRNA isolation/amplification, and microarray hybridizations. VRI provided the construction of the microarrays and tools for the statistical analysis. MWC and EMM conceived the study and participated in its design and helped draft the manuscript. All authors read and approved the final manuscript.Benjamini-Hochberg multiple hypothesis correction (p < 0.01) and the SVD results, along with a 2-fold minimum expression change, was used to identify 140 differentially expressed genes. 19 genes marked with an (*)were also reported in the cervical cancer literature. 30 genes marked with (**)were homologs that were reported in the previous cervical cancer literature.The list of 140 genes differentially regulated between normal cervix and invasive cervical cancer. The intersection of a t-test using the Click here for fileCell line culture conditions. Cell lines were cultured as monolayers in ATCC recommended media. Also, SiHa and HeLa cells were cultured in DMEM as well as in an organotypic environment.Click here for filePotential biomarkers derived from cell lines cluster normal cervix from cervical cancer in a manner comparable to the tissue-derived expression profiles. We identified a cell line with the highest correlation to cervical cancer (C4-I) and generated a list of 196 differentially expressed genes between the C4-I and primary normal cell lines. This list was used to hierarchical cluster the normal cervix and cervical cancer biopsies. The resulting dendrogram was similar to the dendrogram in Figure Click here for fileJNK cascade is a well modeled pathway of cervical cancer by most cell lines whereas the RNA processing pathway is poorly modeled by most cell lines. A detailed view of two specific pathways indicate some pathways are well represented by many cell lines , whereas other pathways  are poorly modeled by many cell lines. Missing data indicates significance was not reached for a particular cell line and not a correlation of -0.4.Click here for filePresentation of the day of hybridization, microarray print batch (written "HS-print batch-slide number"), and dendrogram. In order to reduce the chance of non-biological artifacts that may result in high correlations between samples, replicates for the most part were hybridized on different days. In addition, many samples, including tissue biopsies, were run on different print sets and different days. Additional file Click here for filePercent of SVD component variance. The first 3 components plotted in Figure Click here for file


It is unclear whether insulin-like growth factor (IGF) function is involved in the pathophysiology of chronic fatigue syndrome (CFS). Unpublished data and reports in patient organization newsletters suggest that Acclydine, a food supplement, could be effective in the treatment of CFS by increasing biologically active IGF1 levels. Here we aimed to measure the IGF1 and IGF binding protein (IGFBP) 3 status of CFS patients compared to age- and gender-matched neighborhood controls, and to assess the effect of Acclydine on fatigue severity, functional impairment, and biologically active IGF1 level (IGFBP3/IGF1 ratio).A randomized, placebo-controlled, double-blind clinical trial.Radboud University Nijmegen Medical Centre, The Netherlands.Fifty-seven adult patients who fulfilled the US Centers for Disease Control and Prevention criteria for CFS. IGF status of 22 CFS patients was compared to that of 22 healthy age- and gender-matched neighborhood control individuals.Acclydine or placebo for 14 wk.Outcomes were fatigue severity , functional impairment (Sickness Impact Profile-8 [SIP-8]), and biologically active IGF1 serum concentrations. Analyses were on an intention-to-treat basis.p = 0.70), SIP-8 +59.1 , and IGFBP3/IGF1 ratio −0.5 .There was no difference in IGF status in 22 CFS patients compared to healthy age- and gender-matched control individuals. Treatment with Acclydine did not result in significant differences compared with the placebo group on any of the outcome measures: CIS-fatigue +1.1 . The other primary outcome measure was “functional impairment,” which examines how well someone carries out their daily life, using the Sickness Impact Profile (SIP-8) scale. The secondary outcome measures included physical activity levels, day-to-day fatigue levels, and the levels of insulin-like growth factor 1 (IGF1) in blood, a hormone that's thought by some researchers to be related to the severity of chronic fatigue.What the trial shows: In the trial, 57 people were randomized to receive either Acclydine plus amino acid supplements, or placebo tablets. When comparing scores on the primary outcome measures at the end of the trial, the researchers did not see significant differences between the treatment and placebo groups. Similarly, the results for secondary outcome measures in this trial did not show any significant differences between the treatment and placebo groups.Strengths and limitations: Strengths of this study include the fact that it is one of the few properly designed studies of a product for which claims have been made of effectiveness in chronic fatigue syndrome. The study was designed as a double-blind trial, in which participants and investigators (those collecting the outcome data) did not know whether an individual received Acclydine or placebo. This procedure should help to minimize bias in assessing outcomes. A key limitation of this study is the relatively small number of participants. However, the investigators considered it unlikely that a larger trial would detect a clinically meaningful effect of Acclydine on these outcome measures for patients with chronic fatigue syndrome.Contribution to the evidence: Systematic reviews of interventions for the treatment of chronic fatigue syndrome have found evidence for the efficacy of exercise therapy and cognitive behavioral therapy. This trial shows no evidence of efficacy of Acclydine plus amino acid supplements for the treatment of chronic fatigue. Chronic fatigue syndrome (CFS) is a medically unexplained syndrome, characterized by severe disabling fatigue for a period of at least 6 mo that has led to considerable impairment in daily functioning . VariousNeuroendocrinological investigations have tried to elucidate the pathophysiology of CFS . As CFS Solanum dulcamara. It has been claimed that Acclydine is effective because it increases IGF1 concentrations in CFS patients by stimulating GH releasing hormone and, consequently, GH secretion. GH is converted to IGF1 in the liver. IGF1 activates tyrosine kinase and integrin receptors and stimulates intracellular lipid and glycogen synthesis.Despite the conflicting publications concerning IGF1 status in CFS patients, in recent years there have been reports in patient organization newsletters that a new food supplement called Acclydine could be an effective treatment for CFS. The active ingredient of Acclydine is an alkaloid from Levels of IGF binding proteins (IGFBPs), particularly IGFBP3, modulate the biological activity of IGF1, and the ratio of IGFBP3 to IGF1 reflects IGF1 biological activity . There ahttp://www.controlled-trials.com).However, there are, to our knowledge, no published peer-reviewed studies investigating the effect of Acclydine. Double-blind randomized controlled trials are needed, for several reasons. First, this food supplement is available on the Internet without prescription and is thus easily accessible. Second, although the effects of Acclydine treatment have not been evaluated thoroughly, there are ongoing studies in other vulnerable patient categories, such as in cancer patients with chronic fatigue after treatment, but these studies are not listed in the international controlled trial registry Current Controlled Trials  criteria for CFS ) or functional impairment  compared to the placebo group.No significant differences in change scores were found between the treatment and placebo groups on the primary outcome measures . The CFSActometer activity scores did not show significant differences between the two groups. Analysis of fatigue severity rated with the DOF did not show significant differences either.No significant differences were found between the treatment and placebo groups in IGF1 blood level, IBFBP3 blood level, or IGFBP3/IGF1 ratio at baseline; after 4, 8, or 14 wk of treatment; or during follow-up .In this randomized controlled blinded clinical trial, no therapeutic effect of Acclydine in CFS was found. In this study we also addressed the issue of a deficiency of bioactive IGF1. To control for unwanted stress effects and other confounders, the blood samples were taken simultaneously from the patients and their matched neighborhood controls. For each pair of patient and control, the measurements of IGF1 and IGFBP3 were performed in the same run. In addition, we detected no differences in IGF1 or IGFBP3 concentrations in CFS patients without psychiatric co-morbidity versus closely matched healthy neighborhood controls.Although the sample size is relatively small, the results do not point to a different IGFBP3/IGF1 status in CFS patients compared to controls. These results do not support the hypothesis that changes in IGF1 and IGFBP3 metabolism are involved in the pathophysiology of CFS.In this study we enrolled a representative sample of adult referred and non-referred CFS patients who fulfilled the international CDC consensus criteria for CFS . In contTo our knowledge, this is the first published report of a randomized controlled trial evaluating the effect of the food supplement Acclydine in CFS patients. The effect was assessed by pre- and post-trial testing with validated instruments designed to assess different dimensions of CFS as well as treatment effects. Treatment with Acclydine was not more effective than placebo in changing self-reported outcome measures, hormonal blood levels, or physical activity.The lack of significant differences on any of the dimensions of fatigue or the hormonal assessments strengthens our overall findings.Previous unpublished studies by others on file at the manufacturer claimed that treatment with Acclydine would be more effective in CFS patients with a higher IGFBP3/IGF1 ratio. Based on this claim, we did a subset analysis for patients with a ratio greater than 4.5:19 CFS patients in the Acclydine group and 20 in the placebo group had a ratio greater than 4.5. No significant differences in change scores were seen between the subgroups in any of the outcome measures.A second claim was that Acclydine treatment works by increasing biologically active IGF1. However, assessment of IGF1 concentration and IGFBP3/IGF1 values in the Acclydine group at baseline, 4 wk, and 8 wk during treatment, at the end of treatment, and at follow-up (1 mo after the treatment period) did not show a significant change.The power of our study was sufficient to detect changes in time in both groups. Thus, the negative findings found in this randomized controlled trial cannot be explained by a power problem. Considering the reported treatment effect on the primary outcome measures, we believe it is very unlikely that a larger trial would detect a clinically meaningful effect.It is of interest to note that we detected a minor placebo effect in this study. This is in accordance with observations from earlier randomized controlled clinical trials in CFS –25. HoweWe did not monitor patient compliance on a daily basis. During the trial, patients had an appointment by telephone or at the outpatient clinic every 2 wk. During these appointments, we assessed whether the capsules were taken as directed, and the treatment protocol for the next 2 wk was discussed with the patient. Although adherence could not be verified fully, there is no reason to believe that lack of adherence can explain the negative findings.In conclusion, this randomized controlled trial did not demonstrate an effect of Acclydine on biologically active IGF1, nor did it demonstrate any benefit in CFS-related outcome measures. Thus, the findings of this clinical trial do not support the use of Acclydine in the treatment for CFS.We feel that the negative results of this trial are important for two reasons: Acclydine is expensive, so patients might be spending a lot of money on an ineffective treatment, and it is also available without prescription on the Internet, making it available to patients potentially without a doctor's oversight.CONSORT ChecklistClick here for additional data file.(58 KB DOC)Trial ProtocolClick here for additional data file.(262 KB DOC)Alternative Language Abstract S1(12 KB DOC)Click here for additional data file.Alternative Language Abstract S2(162 KB PDF)Click here for additional data file.


To date, sorafenib, a multiple tyrosine kinase inhibitor, is the only systemic agent approved by the FDA in the first-line treatment of patients with unresectable hepatocellular carcinoma (HCC). Several other tyrosine kinase-inhibiting agents have been investigated in the first-line setting, either alone  or in combination with sorafenib (erlotinib and doxorubicin) in phase 3 trials. However, none of these studies demonstrated an improvement in survival over sorafenib. Many agents have also been tested in patients with HCC whose disease has progressed on sorafenib, but regorafenib is the only one to have demonstrated efficacy in this setting in a randomized, phase 3 trial. There were no clear survival benefits shown with everolimus, brivanib, or ramucirumab as second-line therapy. Nivolumab has also shown promising efficacy in patients with HCC who progressed on sorafenib, which was recently granted approval by the FDA, although larger confirmative trials may be considered. The treatment landscape for patients with advanced unresectable hepatocellular tumors has remained fairly static for the past 10 years, with multiple failed trials yield little change in the way these patients might be treated. However, recent findings for regorafenib, lenvatinib, and nivolumab have led to the most significant changes in the treatment paradigm in years. The incidence rate of cancers of the liver and bile duct, of which hepatocellular carcinoma (HCC) is the most common type, has increased over the past 10 years . These sHepatocellular tumors represent a complex set of etiologies that can impact prognosis . MultiplImportantly, the incidence of these different risk factors varies around the world, which impacts surveillance and treatment programs in different countries. For example, patients in the USA are more likely to have NASH while patients in China are more likely to have HBV infection . In addiThus, patients with HCC represent a heterogenous group with a variety of etiologies and a range of presentations. Disease severity plays a critical role in selecting therapeutic regimens for this population given the range of treatments available. The tumor size and location, spread outside of the liver, and vascular invasion all must be taken into consideration when determining the optimal treatment course for any given patient.The complexity of HCC has led to the development of an intricate staging system and set of management guidelines. The Barcelona Clinic Liver Cancer (BCLC) staging system is commonly used to guide the treatment of patients with HCC through a set of validated treatment recommendations based on the extent of a patient’s disease . AccordiEven with the benefits provided by the BCLC staging system, further refinement is needed to address its limitations and improve the treatment of patients with HCC. For example, patients may be classified as Child-Pugh A or B but have a worse prognosis due to other clinically important events, such as episodes of spontaneous bacterial peritonitis, variceal bleeding, hyponatremia, renal dysfunction, or malnutrition, which are not captured by this classification. Patients may also be classified as Child-Pugh C but satisfies the Milan criteria and therefore should be considered for liver transplant instead of best supportive care. Future efforts to refine the BCLC staging system should also incorporate molecular profiles and biomarkers into the current algorithm to more effectively guide clinical decision-making. In addition, greater knowledge of the impact of patterns of progression and development of adverse events (AEs) on treatment and survival is needed.Sorafenib, which is recommended for patient with BCLC C status, is the first systemic therapy to demonstrate efficacy in patients with unresectable HCC  and has The SHARP trial was the first study to demonstrate efficacy of a systemic treatment for patients with unresectable HCC . In thisP < 0.001; Table The results showed a significantly longer median overall survival (OS) in the sorafenib group relative to the placebo group  [Further support for the use of sorafenib in this population was provided by an additional phase 3 trial conducted in patients from the Asia-Pacific region . The res= 0.014) , 26. In = 0.014) , lendingP = 0.0014), a difference that was consistent across all pre-planned stratification groups. In addition, progression-free survival (PFS) and TTP both did not differ between arms. Grade 3 or 4 AEs occurred in 432 patients (82.1%) in the sunitinib arm and 402 patients in the sorafenib arm (74.2%). Patients on sorafenib were more likely to have dose reductions due to AEs (35.1 vs. 30.0%), but patients on sunitinib were more likely to have temporary discontinuations of treatment (76.6 vs. 58.7%).Additional phase 3 studies have been conducted to examine other novel first-line treatments; however, to date, all have failed to meet their primary efficacy end points. Sunitinib, an inhibitor of multiple tyrosine kinases, was compared with sorafenib in 1074 patients with HCC who had no prior systemic treatment . This trOther novel small molecules have been compared with sorafenib in first line, including the vascular endothelial growth factor (VEGF) and fibroblast growth factor receptor inhibitor brivanib in the BRISK-FL study . This stThe pattern of results was slightly altered when the VEGF and platelet-derived growth factor receptor (PDGFR) inhibitor linifanib was compared with sorafenib in 1035 patients with HCC in the LIGHT study . ConsistP = 0.408). TTP also did not differ significantly between the combination and single-agent arms. In contrast to these end points, DCR was significantly higher in the single-agent sorafenib arm (52.5%) than in the group treated with sorafenib + erlotinib (43.9%), although it is important to note that treatment duration in the sorafenib arm was longer than in the combination arm (123 vs. 86 days) and therefore the longer duration of DCR could be attributed to the longer treatment with the single agent. Overall AE profiles were similar between the two treatment groups, although rash/desquamation, anorexia, diarrhea, and nose bleeding were more likely to occur in the group that received the combination of sorafenib + erlotinib.Investigations in first line have not been limited to single-agent regimens; the SEARCH study assessed the efficacy of sorafenib with or without erlotinib, an epidermal growth factor receptor inhibitor, for patients with HCC . Median The cytotoxic chemotherapy agent doxorubicin had been used as the systemic therapy agent for HCC without the support of a randomized study for many years before sorafenib was approved. The combination of doxorubicin with sorafenib was investigated as first-line treatment for patients with HCC in the CALGB 80802 study . The stun = 476). Although OS was not significantly longer, some secondary endpoints favored lenvatinib over sorafenib, including PFS (7.4 vs. 3.7 months) and TTP (8.9 vs. 3.7 months). Median duration of treatment was 5.7 months in the lenvatinib arm and 3.7 months in the sorafenib arm; however, 13% of lenvatinib-treated patients discontinued treatment due to AEs compared with 9% of sorafenib-treated patients.Most recently, the trial assessing non-inferiority of the PDGFRα, RET, and KIT inhibitor lenvatinib relative to sorafenib could potentially be considered a success, although the trial was not designed to determine if OS was significantly improved by lenvatinib . In the N = 395), a large majority of the patients (86%) randomized to brivanib or placebo had progressed on sorafenib; the remainder were sorafenib intolerant. Median OS, the primary end point, was not significantly different between the brivanib (9.4 months) and the placebo (8.2 months) arms, even when post-study treatment was considered. However, TTP was significantly longer for patients on brivanib than on placebo (4.2 vs. 2.7 months). Discontinuation due to AEs  was reported in 23% of patients receiving brivanib.Options for patients who progress on sorafenib have been limited given that there were no treatments with demonstrated efficacy and safety in large phase 3 trials prior to regorafenib. In addition to the study in first line, brivanib was also investigated in patients who progressed on sorafenib in the BRISK-PS study . In thisEverolimus, a mammalian target of rapamycin inhibitor, was investigated in patients with HCC who progressed on or were intolerant to sorafenib in the EVOLVE-1 study . Five huInvestigations into treatments for patients who have progressed on sorafenib have not been limited to oral agents; the REACH study assessed the intravenously administered anti-VEGFR-2 antibody ramucirumab in this population . SimilarThe JET-HCC study was designed to assess the benefits of the c-MET inhibitor tivantinib in a subset of the second-line HCC population . PatientP < 0.0001). PFS  and TTP  were also significantly longer with regorafenib. Patients on regorafenib also had a higher ORR (11 vs. 4%) and DCR (65 vs. 36%) relative to patients on placebo. All patients treated with regorafenib and 93% of patients on placebo experienced treatment-emergent AEs during the study. Most adverse events were grade 1 or 2. The most common grade 3 or 4 AEs (regorafenib vs. placebo) were hypertension (15 vs. 5%), HFSR (13 vs. 1%), fatigue (9 vs. 5%), and diarrhea (3 vs. 0%). Grade 5 AEs  occurred in 13% of regorafenib patients and 20% of placebo patients.To date, the multikinase inhibitor regorafenib is the only agent to have demonstrated safety and significant efficacy as a second-line systemic therapy in a large, randomized phase 3 study . The RESN = 214 in the dose expansion phase) [Immuno-oncology may represent a promising new direction for the treatment of patients with unresectable HCC. The checkpoint inhibitor nivolumab was the second agent to show clinical benefits for patients with HCC in the international, non-comparative, open-label phase 1/2 CheckMate-040 study (n phase) . This trAlthough this trial was not randomized, the results were considered favorable enough to warrant conditional approval by the FDA for nivolumab in the treatment of patients with HCC who have progressed on sorafenib. Further research from larger, randomized trials Table  will be Understanding the potential reasons behind the success or failure of clinical trials for patients with advanced HCC can provide a useful roadmap for future developments for this population of patients with limited treatment options. Potential reasons for the failure of some of these agents include, but are not limited to, a lack of efficacy based on their mechanisms of action, the clinical heterogeneity/pathogenesis of HCC and the absence of established biomarkers capable of predicting outcomes. Also, trials in first line may be insufficiently powered to show advantages of investigational agents relative to sorafenib given that most of the agents that were evaluated were anti-angiogenic multikinase inhibitors sharing some common pathways Table . FailureLlovet and Hernandez-Gea have suggested that sorafenib provides a clinical benefit to patients with HCC because it balances the targeting of tumor cells and their microenvironment with a manageable toxicity profile . NotablyTrial design may also play a critical role in the success or failure of clinical trials in HCC. Underlying disease characteristics, such as the extent of cirrhosis and hepatitis, that are defined in the enrollment criteria for each trial may influence the results, and future trials should be balanced with regard to these factors. In addition, further research into prognostic factors in failed trials as well as those that have succeeded should provide a better understanding of just how disease characteristics can contribute to trial outcomes. To that end, the design of the RESORCE trial may have helped facilitate the positive outcomes for regorafenib. The carefully defined criteria for prior sorafenib exposure ensured that patients were randomized to regorafenib after showing initial tolerability to sorafenib and may have improved the tolerability of regorafenib for those who enrolled. The additional requirement of randomization no more than 10 weeks after discontinuation of sorafenib should have limited progression in patients who enrolled in the trial .The selection criteria for RESORCE also specified that all patients had good PS, thereby ensuring that all patients would be able to tolerate treatment . Indeed,A look at the history of phase 3 trials for patients with HCC shows that, until the recent trials of regorafenib and nivolumab, that improving survival in this population is a high bar to pass. Numerous drugs with different mechanisms of action have attempted to demonstrate efficacy in this population and failed to meet their end points. Interestingly, many of these agents showed improvements in TTP and PFS that did not translate into prolonged OS, calling into question the clinical relevance of these end points in HCC. More data on symptomatic progression and quality of life would be helpful for determining the role of these end points in future trials.Additional targeted and immuno-oncology agents are also currently under development and results from these trials and others have the potential to impact the future of treatment for patients with HCC. For example, ongoing research will assess the benefits of tivantinib in patients with unresectable HCC that expresses high levels of MET. Also, multiple compounds targeting FGF/FGFR, which may be related to MET signaling, are also in development. Recent results have led to the most significant changes in the treatment landscape for patients with HCC in a decade and this trend is likely to continue as new data are reported.Future developments may also be improved by a better understanding of HCC pathogenesis, for example through genome-wide association studies. Further research into the underlying etiology of hepatocellular tumors may facilitate a more personalized approach to trial design based on biomarker expression. Additional research into how underlying liver diseases  may affect outcomes can also help to personalize treatment selection in HCC although it is important to note that the effects of sorafenib were observed in both patients with HBV or HCV in pooled dataset from the randomized SHARP and Asia-Pacific trials .Despite previous failures, promising new developments are on the horizon for patients with HCC. Improved treatments for hepatitis may limit its contribution to HCC pathogenesis, reducing the number of cases and altering the disease characteristics of future patients with HCC such that different treatment approaches may be warranted. Immunotherapy may also play an important role in the future treatment of patients with HCC. The anti-PD-1 antibodies nivolumab and pembrolizumab as well as the anti-cytotoxic T-lymphocyte associated protein (CTLA)-4 antibody ipilimumab are all currently being investigated in different phases for patients with HCC. These agents could provide new treatment options, either alone or in combination with current TKI therapies, for this patient population.


We have previously developed an approach, where two-dimensional gel electrophoresis (2DE) was used, followed by sectional analysis of the whole gel using high-resolution nano-liquid chromatography-mass spectrometry (ESI LC-MS/MS). In this study, we applied this approach on the panoramic analysis of proteins and their proteoforms from normal (liver) and cancer (HepG2) cells. This allowed us to detect, in a single proteome, about 20,000 proteoforms coded by more than 4000 genes. A set of 3D-graphs showing distribution of these proteoforms in 2DE maps (profiles) was generated. A comparative analysis of these profiles between normal and cancer cells showed high variability and dynamics of many proteins. Among these proteins, there are some well-known features like alpha-fetoprotein (FETA) or glypican-3 (GPC3) and potential hepatocellular carcinoma (HCC) markers. More detailed information about their proteoforms could be used for generation of panels of more specific biomarkers. Today, the main aim in human proteomics is a complete catalogue of all human proteins. As the human genome is deciphered this task becomes clear and purposeful; if there is a protein-coding gene a corresponding protein should be found. Until now, this survey of proteins was performed mainly using a bottom-up approach that includes protein digestion and subsequent mass spectrometric analysis of the peptides produced . The sitv/v) formic acid. MS/MS analysis was carried out in duplicate on an Orbitrap Q-Exactive Plus . Exponentially modified PAI (emPAI) defined as the number of identified peptides divided by the number of theoretically observable tryptic peptides for each protein was used to estimate protein abundance .,18.17,18bundance ,25.We have performed a panoramic study of human proteins and their proteoforms using a cancer cell line (HepG2) and normal liver tissue. Previously, some of these data were already published ,19. We gIn the bottom, the number of proteins (genes) detected by shotgun mass-spectrometry (1221) using FASP protocol (left ellipse (1221): the liver, right ellipse 1467): HepG2 cells) is presented. Only 666 proteins were detected in both liver and HepG2 cells, while 555 proteins were detected only in liver, and 801were detected only in HepG2 cells. This is because of the level of detection sensitivity in our experiment and the levels of proteins in liver and HepG2 cells. The quantity of some proteins is enough to be detected in both samples, but some are only detected in liver and not in HepG2 cells and vice versa. This statement is confirmed by experiments using sectional analysis (top ellipses), when many more proteins were detected. Using sectional analysis, a total of 1920 proteins were detected in both liver and HepG2 cells (including many that were only detected in liver or HepG2 cells before). Again, many proteins were detected in liver only (1385) or HepG2 cells (1853). Concerning sensitivity, it is relevant to stress that only 293 proteins were detected in HepG2 cells but not in liver  using both types of experiments. That confirms our statement about the sensitivity issue. Additionally, it is interesting to compare our data with data published in the paper by Wiśniewski et al. [67: HepG2The main part of this study is a set of proteoform profiles that we generated based on a combination of 2DE with LC ESI-MS/MS. We have produced these profiles as 3D graphical images. Some profiles are very similar in both samples and contain only one or two proteoforms (peaks). Often, proteins have many proteoforms, and the profiles for some of them are very different in liver and HepG2 cells. The most abundant peak usually has pI/Mw coordinates that are congruent with theoretical ones. The profiles of some proteins have an exceptionally large amount of proteoforms. Mostly, these are samples from HepG2 cells. Keeping in mind the cancerous nature of these cells, we have paid special attention to proteins that already are used or are under consideration to be used as tumor biomarkers. It is of note that the list of such markers is actually very long . Since oThe most well-known protein and the only one approved for clinical usage as a marker for HCC is alpha-fetoprotein (FETA) ,28. FETAIn general, profiles of proteins in HepG2 cells tend to have more proteoforms (peaks) than in liver, but opposite cases are also observed. According to the positions of the peaks, preliminary assumptions about their origins can be made. For instance, the lower weight proteoforms can be products of proteolysis, which itself can be of different nature . The proteoforms having similar Mw but more acidic pI, compared to the theoretical pI, can be the products of PTMs that add negative or remove positive charges . The shift to the basic direction can be a result of carboxyl group modifications . Some PTMs leading to a big shift in pI, as well as in Mw, are ubiquitination, SUMOylation, or glycosylation. Splice variation is another reason to produce peaks located differently from the canonical sequence pI/Mw position. Such situations should not have a great impact as we have been showing only proteoforms of the same isoforms in the graphs. Taken together, we cannot specify the type of modifications but only give an estimation (by pI/Mw) of these proteoforms. For more specific evaluation of proteoforms a detailed analysis of each profile should be performed.There is a resolution challenge in our experiments. On average, we found that every protein profile in liver and in HepG2 cells has five proteoforms. It is necessary to remind the reader that a single PTM, like acetylation, methylation or phosphorylation can produce a pI shift close to 0.05 ,45. HoweA combination of top-down proteomics (2DE separation of proteins) with bottom-up proteomics allows very convenient visual representations of information about diverse proteoforms coded by the same genes (proteoform profiles). Here, we have compared more than 1100 profiles of the most abundant proteins in liver and HepG2 cells. Among them, we analyzed profiles of known and potential HCC markers that could be helpful to further improve the specificity of testing for this disease. In case of alpha-fetoprotein (FETA), we found its presence in HCC as multiple proteoforms. There is a chance that among these there could be forms more specific than AFP-L3. Profiles of heat shock protein beta 1 (HSPB1) and annexin 2 (ANXA2) in HepG2 compare to liver cells are characterized by pronounced shifts of proteoforms towards more acidic pIs. It usually happens in case of phosphorylation or acetylation, and these proteins are known for these PTMs. Accordingly, these proteoforms could be better HCC markers than the levels of HSPB1 and ANXA2.