Autism is a behavioral disorder with impaired social interaction, communication, and repetitive and stereotypic behaviors. About 5–10 % of individuals with autism have 'secondary' autism in which an environmental agent, chromosome abnormality, or single gene disorder can be identified. Ninety percent have idiopathic autism and a major gene has not yet been identified. We have assessed the incidence of chromosome abnormalities and Fragile X syndrome in a population of autistic patients referred to our laboratory.Data was analyzed from 433 patients with autistic traits tested using chromosome analysis and/or fluorescence in situ hybridization (FISH) and/or molecular testing for fragile X syndrome by Southern and PCR methods.The median age was 4 years. Sex ratio was 4.5 males to 1 female [354:79]. A chromosome (cs) abnormality was found in 14/421 [3.33 %] cases. The aberrations were: 4/14 [28%] supernumerary markers; 4/14 [28%] deletions; 1/14 [7%] duplication; 3/14 [21%] inversions; 2/14 [14%] translocations. FISH was performed on 23 cases for reasons other than to characterize a previously identified cytogenetic abnormality. All 23 cases were negative.Fragile-X testing by Southern blots and PCR analysis found 7/316 [2.2 %] with an abnormal result. The mutations detected were: a full mutation (fM) and abnormal methylation in 3 [43 %], mosaic mutations with partial methylation of variable clinical significance in 3 [43%] and a permutation carrier [14%].The frequency of chromosome and fragile-X abnormalities appears to be within the range in reported surveys (cs 4.8-1.7%, FRAX 2–4%). Limitations of our retrospective study include paucity of behavioral diagnostic information, and a specific clinical criterion for testing.Twenty-eight percent of chromosome abnormalities detected in our study were subtle; therefore a high resolution cytogenetic study with a scrutiny of 15q11.2q13, 2q37 and Xp23.3 region should be standard practice when the indication is autism. The higher incidence of mosaic fragile-X mutations with partial methylation compared to FRAXA positive population [50% vs 15–40%] suggests that faint bands and variations in the Southern band pattern may occur in autistic patients. Autism, often referred to as autistic disorder or infantile autism, is a complex behavioral disorder, which, by definition, develops prior to age three. Autism is defined completely on the basis of impairments in social interaction and communication, repetitive and stereotypic behaviors. Recent research has examined autistic traits in a population of twins and found that social impairment actually follows a unimodal distribution without a clear demarcation to separate cases of the disorder [In 1943 Kanner coined "infantile autism" , is the primary diagnostic reference used in the United States for autism. The causes of autism can be divided into "idiopathic," which comprises the majority of cases, and "secondary," in which an environmental agent, chromosome abnormality, or single gene disorder can be identified. About 5–10% of individuals with autism can be diagnosed with secondary autism; the remaining 90–95% have idiopathic autism. About 30% of children with idiopathic autism have complex autism, defined by the presence of dysmorphic features or microcephaly or a structural brain malformation [A recent CDC case-finding study in Brick Township, New Jersey reported prevalence at 40 per 10,000 for autism . The sexSocial cognition and communication in autism may be related to dysfunction in the amygdala, hippocampus, and related limbic and cortical structures. The cerebellum may also form part of a distributed neuronal network responsible for social cognition and communication. Serotonin is the neurotransmitter implicated in autism The single gene disorders in which secondary autism is observed include fragile X syndrome, tuberous sclerosis, phenylketonuria, Rett syndrome ,9, SotosTo gain further insight into secondary autism we have compiled data on patients with autistic traits tested for fragile-X syndrome using molecular methods and chromosome abnormalities using cytogenetic analysis and FISH.A search was initiated using the key word autism in the indication field of the Genzyme Genetics, Orange laboratory database. For each case the electronic data and/or files were reviewed. The referral center, age, sex, karyotype, fluorescence in situ hybridization (FISH) and fragile X results were extracted and tabulated.G-banded metaphases were prepared using standard procedures and FISH was performed using the protocol provided by the manufacturer of the commercial probes (Vysis Inc., Illinois, MI).Fragile X test: Isolated DNA was tested by both Southern blot analysis and Polymerase chain reaction (PCR) for the size and methylation status of the CGG repeat expansion within the FMR-1 gene. Southern blot analysis was performed with the probe StB12.3 on EcoR1 and Eag1 digested DNA. PCR products were separated by acrylamide gel electrophoresis and detected with a CGG repeat probe.Patients with autistic traits were referred by physicians to our laboratory for genetic testing. The clinical diagnostic criteria applied were not specified on the test request forms.A total of 433 patients with an indication of autism were sent to our laboratory for genetic diagnosis. The median age was 4 years. Sex ratio was 4.5 males to 1 female [354:79]  and in other surveys, the common recurring chromosomal abnormality was duplication of the proximal 15q region  receptor subunits that map to the distal, apparently nonimprinted segment of the duplicated region ) ,45. It aulations -31, otheulations -49.Yardin et al 2002  recommenA deletion of 2q37.3 was identified by FISH in a patient with autism and macrocephaly in our study. Wolff et al 2002  also repA de novo deletion of chromosome 3, del(3)(p25), was found in one case with ASD and development delay in our patient pool. A deletion of 3q region was found by Konstantareas & Homatidis 1999 . Genome To date, there have been no reports of 12q deletions in patients with ASD, to the best of our knowledge. For the first time we report an interstitial deletion 46,XY,del(12)(q21.2q23.3) in a patient with ASD, development delay, mental retardation, multiple congenital abnormality, and family history of Down syndrome.46,XY,del(13)(q13.2q14.1)de novo was found in one of our patients' with ASD. Hyperserotonemia in autism is one of the longest-standing biochemical findings. The serotonin 2A receptor gene (HTR2A) on chromosome 13q14q21 is a primary candidate gene in autism. Converging data from recent genome screens also implicates the genomic region containing HTR2A -66. CorrThe recent physical mapping of the serotonin 5-HT(7) receptor gene (HTR7) to 10q23  raises tSo far there has been no observed association or link between chromosome 14 and ASD. Also, the mosaic inversion inv(14)(q11.2q33) [3/20] found in one of our patients is considered a cultural artifact when seen in 1or 2 cells.In this study, a patient with inv(17)(q23q25)de novo, had ASD, hypotonia and developmental delay. Chromosome 17q shows association with autism by genome wide scans and in linkage studies ,66,68. TWe report a patient with 18p deletion due to an unbalanced translocation between 14 and 18. Majority of the reported cases with autism involve deletion of 18q -72. A deOne of the breakpoints in the balanced translocation, 46,XX,t was 1q25, in the present study. A recent report, links D1S1675 that maps to chromosome 1q24 with autism  using obAlthough no cases with X- rearrangements were identified, in this study, the literature on abnormal X chromosome and autism is discussed as it has been cited in multiple cases. An autistic (ICD-10) woman had a translocation, t , a boy wThe typical clinical picture in FRAXA includes mental retardation, macro-orchidism, large ears, and prominent jaw. Within neurons, the FMR protein (FMRP) interacts with mRNA and ribosomes, suggesting a role in regulating protein synthesis . FMRP isA multicenter study in Sweden  found frForty-three percent of FRAX patients had a mosaic mutation in our study. The prevalence of males who carry a full mutation and a permutation is 15–20% -102 amonThe degree of mental retardation seemed not to be influenced by the presence of premutation alleles in some of the cells and a full mutation in the rest of the cells . While MThe molecular mechanism of the FRAX is based on the expansion of a CGG repeat in the 5' UTR of the FMR1 gene in the majority of fragile X patients. The instability of this CGG repeats containing region is not restricted to the CGG repeat itself but expands to the flanking region as well. de Graaff et al 1996  describeA 6-year old mosaic permutation female carrier had 29,65,80,39 repeats. The autism spectrum disorder in our patient may be due to diminished translational efficiency in FMRP production. Tassone et al (2000)  have sh,116,117.The significance of intermediate alleles found in 7/316 (Average age 4.36 years) of our patients requires further exploration on larger independent samples as they may raise the threshold for important developmental disabilities and/or physical features .The limitations of our retrospective study were a lack of uniform clinical criterion for inclusion and limited behavioral diagnostic information for purposes of dissecting the phenotype. However, for autism diagnosis it may be reliable, as shown in a recent study  which exIn our experience, the incidence of chromosome (3.33%) and fragile-X (2.2%) abnormalities totals to 5.53% in a population of patients with an indication of autism sent for genetic testing. Since, 28% percent of chromosome abnormalities detected in our study were subtle; a high resolution cytogenetic study for ASD patients with a scrutiny of 15q11.2q13, 2q37 and Xp23.3 region should be standard practice. The higher incidence of mosaic fragile-X mutations with partial methylation in our ASD population vs incidence of mosaicism in reported populations with fragile X syndrome [50% vs 15–40%], suggests that faint bands and variations in the Southern band pattern may occur rather frequently in fragile X positive autistic patients. The mosaic FRAXA mutation with normal and abnormal or partial methylation may also enhance the threshold for autism spectrum disorder. Careful analysis and high quality gels are required to rule out the type of mutation in patients with autistic traits. FRMP estimates in positive cases would be an extremely useful adjunct especially in mosaic cases. Future studies are necessary to corroborate the high incidence of mosaicism and their role in ASD.The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


Intra-aortic balloon counterpulsation (IABC) has an established role in the treatment of patients presenting with critical cardiac illnesses, including cardiogenic shock, refractory ischemia and for prophylaxis and treatment of complications of percutaneous coronary interventions (PCI). Patients requiring IABC represent a high-risk subset with an expected high mortality. There are virtually no data on usage patterns as well as outcomes of patients in the Indo-Pakistan subcontinent who require IABC. This is the first report on a sizeable experience with IABC from Pakistan.Hospital charts of 95 patients (mean age 58.8 (± 10.4) years; 78.9% male) undergoing IABC between 2000–2002 were reviewed. Logistic regression was used to determine univariate and multivariate predictors of in-hospital mortality.The most frequent indications for IABC were cardiogenic shock (48.4%) and refractory ischemia (24.2%). Revascularization  was performed in 74 patients (77.9%). The overall in-hospital mortality rate was 34.7%. Univariate predictors of in-hospital mortality included (odds ratio [95% CI]) age (OR 1.06 [1.01–1.11] for every year increase in age); diabetes (OR 3.68 [1.51–8.92]) and cardiogenic shock at presentation (OR 4.85 [1.92–12.2]). Furthermore, prior CABG (OR 0.12 [0.04–0.34]), and in-hospital revascularization (OR 0.05 [0.01–0.189]) was protective against mortality. In the multivariate analysis, independent predictors of in-hospital mortality were age (OR 1.13 [1.05–1.22] for every year increase in age); diabetes (OR 6.35 [1.61–24.97]) and cardiogenic shock at presentation (OR 10.0 [2.33–42.95]). Again, revascularization during hospitalization (OR 0.02 [0.003–0.12]) conferred a protective effect. The overall complication rate was low (8.5%).Patients requiring IABC represent a high-risk group with substantial in-hospital mortality. Despite this high mortality, over two-thirds of patients do leave the hospital alive, suggesting that IABC is a feasible therapeutic device, even in a developing country. Intra-aortic balloon counterpulsation (IABC) has an established role in the treatment of patients presenting with cardiogenic shock -3, refraWe reviewed the charts of 95 patients undergoing IABC at the Aga Khan University Hospital (AKUH), Karachi, Pakistan between January 2000 and December 2002. Patients requiring IABC in the operating room immediately following CABG to assist weaning off cardiopulmonary bypass were excluded from this study. However, those patients who underwent IABP implantation prior to surgery were included. The AKUH is a tertiary care hospital located in the metropolitan city of Karachi that receives a mixture of affluent as well as low and middle income patients and serves the entire city as a referral center for patients requiring high-intensity tertiary care. Variables collected included age, gender, indication for IABC (shock or non-shock), history of diabetes, hypertension, smoking, prior PCI or CABG, left ventricular function, refractory ischemia and treatment (revascularization vs. no revascularization). Cardiogenic shock was defined as a systolic blood pressure (SBP) of < 90 mm Hg for at least 30 minutes (or requirement of inotropes to maintain a SBP > 90 mm Hg) associated with hypoperfusion (decreased urine output or cool extremities) and a heart rate of ≥ 60 beats per minute. Left ventricular (LV) ejection fraction (EF) was assessed by visual estimation. LV function was recorded as normal for an EF of ≥ 55%, mildly impaired for an EF 40–54%, moderately impaired for an EF 26–39% and severely impaired if the EF was ≤ 25%. Heart failure was diagnosed using clinical signs as defined by the Framingham criteria . RefractAll variables were entered into Statistical Package for Social Sciences (SPSS) version 10. Means and standard deviations were calculated for continuous variables and frequencies for categorical variables. Variables were analyzed by simple logistic regression to calculate the unadjusted odds ratios for factors associated with in-hospital mortality. Those variables with a p value of ≤ 0.25 on univariate analysis were entered into the multivariable model and adjusted odds ratios for factors associated with in-hospital mortality were calculated. Finally, the model fit was assessed using the Hosmer-Lameshow test. A p value of < 0.05 was considered significant.Table Table 2 6.09; p = 0.637). In the adjusted analysis, a prior history of CABG did not remain a significant predictor of survival primarily because forty-five out of 46 patients underwent revascularization.In the multivariate analysis Table , the sigWhen age as a risk factor was further analyzed by plotting an ROC curve, an age cut-off of 66.5 years had a high specificity for the outcome of in-hospital mortality . Thus older patients requiring IABC suffer worse outcomes than younger subjects.The overall complication rate related to the device implantation was low. Eight patients (8.5%) developed limb ischemia necessitating removal of the IABP; however, only one of these eight required surgery. There were no significant bleeding complications although one patient developed a hematoma following removal of the device; however this patient did not require blood transfusion or surgical repair of the arteriotomy site.Patients requiring IABC are at high risk for death on account of their critical underlying conditions. Despite this, several data have suggested that IABC can improve morbidity and mortality in specific subsets of patients including those presenting with cardiogenic shock. The use of IABC in developing countries is limited on account of lack of equipment as well as skilled personnel who can insert and manage the device. Ours is the first report on a sizeable experience with IABC from the Indo-Pakistan subcontinent. Our experience is similar to that of other centers in the West. We report a high in-hospital mortality rate in patients undergoing IABC . However, given that nearly half of the subjects had cardiogenic shock at presentation, this mortality rate is reasonably acceptable. Advanced age (over 66.5 years), diabetes and cardiogenic shock at presentation were strong independent predictors of in-hospital mortality, while revascularization  was associated with high odds of survival. The latter finding is consistent with recently reported data from the IABP Benchmark Registry . Of partSeveral limitations of this study should be acknowledged. First, the sample size is fairly small and this is reflected in the relatively wide confidence intervals for the odds ratios. Due to a small sample size, it is difficult to make a comparison of correlates of mortality between subgroups, for example those presenting with cardiogenic shock versus those who did not and those undergoing surgical versus percutaneous revascularization. However, as expected, patients presenting with shock had a significantly higher mortality . Second, the patient group selected may not be representative of other centers in Pakistan given that our institution is a unique tertiary care hospital in the country. Third, our cohort did not contain patients undergoing prophylactic IABC prior to high-risk PCI for indications other than cardiogenic shock. This probably represents practice patterns at our institution whereby, largely due to cost constrains, very high-risk patients (for example those with multivessel disease and/or severe impairment of LV function) are preferentially send for surgery. The cumulative cost of IABC with multivessel stenting far exceeds that of a bypass operation. Only two patients required emergent IABC during PCI in the study period. This may reflect a selection of lower risk patients for PCI at our institution. Fourth, while the survival rate following IABC is nearly 65%, no analysis has been made of the cost effectiveness of this therapy.In conclusion, cardiogenic shock and refractory ischemia are common indications for IABC in a Pakistani setting. Patients requiring an IABP represent a high-risk group with substantial in-hospital mortality. This is consistent with the nature of the presenting illnesses in these patients and is similar to western data. Despite this high mortality, over two-thirds of patients do leave the hospital alive, suggesting that IABC is a feasible therapeutic device, even in a developing country. Age (particularly over 66.5 years), diabetes and cardiogenic shock at presentation are significant predictors of mortality in this group of patients. Revascularization is a significant predictor of survival and complication rates are acceptably low. Larger studies are needed to evaluate which subsets of patients benefit the most from this device and further cost effectiveness analyses are warranted.The author(s) declare that they have no competing interests.FHJ, SAK conceptualized this study and participated in the study design. FHJ performed the statistical analysis. HK, NFM collected the data. KAK, SD, AS were involved in manuscript review. AH, JT and NN participated in manuscript drafting and review. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Between cell divisions the chromatin fiber of each chromosome is restricted to a subvolume of the interphase cell nucleus called chromosome territory. The internal organization of these chromosome territories is still largely unknown.We compared the large-scale chromatin structure of chromosome territories between several hematopoietic chicken cell types at various differentiation stages. Chromosome territories were labeled by fluorescence in situ hybridization in structurally preserved nuclei, recorded by confocal microscopy and evaluated visually and by quantitative image analysis. Chromosome territories in multipotent myeloid precursor cells appeared homogeneously stained and compact. The inactive lysozyme gene as well as the centromere of the lysozyme gene harboring chromosome located to the interior of the chromosome territory. In further differentiated cell types such as myeloblasts, macrophages and erythroblasts chromosome territories appeared increasingly diffuse, disaggregating to separable substructures. The lysozyme gene, which is gradually activated during the differentiation to activated macrophages, as well as the centromere were relocated increasingly to more external positions.Our results reveal a cell type specific constitution of chromosome territories. The data suggest that a repositioning of chromosomal loci during differentiation may be a consequence of general changes in chromosome territory morphology, not necessarily related to transcriptional changes. It is a longstanding observation that chromatin distribution in the interphase cell nucleus varies with the cell type. Flemming described differences in nuclear appearance in 1882 . However-7], the We amended our experimental approach with the determination of the position of a gene locus relative to its chromosome territory. In several previous studies it was observed that a number of active genes located preferentially at the surface of their chromosome territories or even outside -12, whilcLys), which is highly active in macrophages, as a model system to explore the possibility of positional changes during activation of a single gene. cLys does not have co-regulated neighbors. Recently the gene cGas41 was found only 200 bp downstream of the polyA-site of cLys. cGas41 is expressed on a low level in all chicken tissues and cell lines tested, including all cell lines used here [cLys regulation were greatly facilitated by cell lines representing these differentiation stages [So far, a correlation of gene activation with increasing looping-out from the chromosome territory has only been shown for gene clusters but not for single genes. In the present study we have chosen the chicken lysozyme gene  and the chromosome 1 centromere relative to their chromosome 1 territory. We found that not only the lysozyme gene domains but also the centromeres were mostly in the chromosome territory interior in multipotent myeloid precursor cells and relocated to the territory periphery in further differentiated cell types. In addition, we determined the radial positioning of the chromosome territories 1 and 8 within the nuclei of each cell type and measured nuclear volumes.In the present study, we investigated the large-scale chromatin organization of chromosome territories in well characterized cell lines representing the five chicken cell types described above Figure . Multi-c3D FISH was performed on formaldehyde fixed, structurally preserved nuclei. Visual examination of painted chromosome 1 and chromosome 8 territories revealed differences between the cell types Figure . In proeA comparison of chromosome territory surface and chromatin texture by visual inspection is bound to subjective influences. To allow an unbiased, quantitative evaluation of chromosome territory morphology, we counted the number of objects to which chromosome territories disaggregate at increasing threshold levels. With a computer program newly developed for this purpose  in 48% of the cases . The difference in distribution between precursor cells and all other cell types was highly significant (p < 0.001) as was the difference between activated macrophages and all other cell types (p < 0.001). The difference between unstimulated macrophages and proerythroblasts (p = 0.003) or myeloblasts (p = 0.024) was also significant whereas the difference between myeloblasts and proerythropblasts was not (p = 0.5). To test the robustness of our results, we repeated statistical analysis after reducing the number of applied categories of localization to only three: internal (A+B), peripheral (C-E) and external (F+G). We confirmed highly significant differences when precursor cells or activated macrophages were compared to any other cell type . In summary, for the lysozyme gene we found a change in position from interior when not expressed in myeloid precursor cells to peripheral when strongly expressed in activated macrophages. This would fit the hypothesis that highly expressed genes are preferentially located in the territory periphery, as it was found previously for large gene clusters [The positions of both, the lysozyme gene domain and the centromere, differed largely between the cell lines representing the various differentiation stages. To classify the positions of the signals, we used the scheme shown in Figure s Figure . In addie cGas41 . We concs Figure  where thclusters ,11. HowecLys gene domain . The same was true for proerythroblasts (p < 0.001) but no significant difference was present in myeloblasts (p > 0.1). These three cell types grow in suspension and have round nuclei. In flat nuclei of LPS-stimulated macrophages we again found chromosome 1 territories more peripheral than chromosome 8 territories (p < 0.005). The radial distribution is also reflected by the signal median values. It indicates at which nuclear radius half of the signal voxels are more internal and half are more external. In 73% of the precursor cells the chromosome 1 signal median is more external than the chromosome 8 signal median. The respective values are 52% for myeloblasts, 82% for proerythroblasts and 91% for activated macrophages.cLys-probe . In unstimulated macrophages the opposite was true (76% vs. 73%, p < 0.05). In the other cell types the differences between the signal medians of cLys and chromosome 1 territories were between 0–2% and not significant.The radial distribution of the lysozyme gene domain did not differ significantly between the cell lines (p > 0.1). The mean value of the signal medians was between 71 and 76% of the nuclear radius for all cell lines. In erythroblasts, chromosome 1 territory signal medians were more internal than Our results are compatible with previous data  in descr3 (± 75 standard deviation, n = 76) and for myeloblasts 327 ± 101 μm3 (n = 50). The difference between the two cell types was highly significant (p < 0.001). The mean nuclear volume of proerythroblast was 296 ± 109 μm3 (n = 78). Proerythroblasts did not consistently show significant differences when compared to precursor cells or myeloblasts. Since the macrophage cell line carries additional chromosomes, its nuclear volume cannot be directly compared to the other cell lines. For unstimulated macrophages we determined nuclear volumes of 459 ± 91 μm3 (n = 42) and for LPS stimulated macrophages 554 ± 139 μm3 (n = 78). This difference was not significant.The volume of nuclei was measured in confocal stacks of DNA counterstain of data sets described above by the same program that was used for the calculation of the radial distributions Figure . The meaThe measured nuclear volume depends on the chosen signal threshold. Since the volume increases by the power of 3 with the nuclear radius, small differences in the segmentation can lead to large volume differences. A cautious interpretation of such measurements is thus advised. The difference between multipotent myeloid precursors and myeloblasts is so large however that we are confident that it is real and not a thresholding artifact.Cell type specific chromatin distributions on the nuclear level have been described for over a century [, p.100].To quantify chromatin dispersal of labeled chromosomes in cells of various differentiation stages, we counted the number of separate, labeled chromatin objects to which the chromosome territories disintegrated at increasing thresholds. In the investigated chicken cell types, chromosome territories of further differentiated cell types disaggregated into more objects. An increase in object number during differentiation may indicate that a significant number of compact chromatin domains with silent genes separate from each other into several, more decondensed, "open" chromatin domains. This would increase the accessibility to transcription factor complexes from the interchromatin compartment by increasing the available chromatin surface area of the chromosome territory. In human lymphoblasts gene rich chromosome 19 territories were found more decondensed than gene poor chromosome 18 territories  and elecMultipotent myeloid precursor cells have the smallest nuclei of the cell types investigated here. Myeloblasts have on average larger nuclei than proerythroblasts. If the observed disaggregation of chromosome territories were based on a nuclear volume increase, the larger myeloblast nuclei should have a stronger dispersion of chromosome territories than proerythroblasts. However, the opposite is true Figure . We thuscLys signal is more exterior than the chromosome 1 territory signal median but in unstimulated macrophages the opposite is true also argues for a cell type specific organization of chromosome territories. A similar observation has been described for a IL-3 induced differentiation of human leukemic K562 cells where the β-globin gene cluster does not change nuclear position but the harboring chromosome 11 territory does [The lysozyme gene domain is positioned inside the chromosome 1 territory in multipotent myeloid precursor cells where the lysozyme gene is inactive, but on the surface or outside in most of the territories in activated macrophages where the gene is strongly expressed. We thus did find a tendency to more exterior regions of the chromosome territory for the highly expressed gene from in activated macrophages although actual looping-out (without visible contact to the territory) was observed in only about 6%. Interestingly, while the radial distribution of the lysozyme gene domain within the nucleus is about the same in all cell types, the harboring chromosome 1 territories show differences. The finding that in erythroblasts the ory does . Howeverory does  or to orory does ,53]. UnfcGas41, which, albeit on a low level, is expressed in all cell types used in this study, including multipotent myeloid precursor cells [While we can exclude a tethering of the inactive lysozyme gene to the centromere, at first glance this result seems compatible with the hypothesis that inactive genes are stored away in internal regions of the chromosome territory and active genes are on their surface or even looped out. However, several aspects suggest an alternative explanation. (i) Embedded in the chicken lysozyme gene domain is a second gene, or cells . Thus weor cells . (ii) Alor cells ] and froor cells . FormallWe describe several features of chromosome territory organization that differ between various hematopoietic chicken cell lines. While multipotent myeloid precursor cells had compact chromosome territories, the more differentiated cell types investigated here displayed somewhat disaggregated, diffuse territories. Although nuclear volumes generally are larger in the more differentiated cell types, they do not correlate with the changes in chromosome territory morphology. The chicken lysozyme locus as well as the chromosome 1 centromere is located preferentially in the interior of the chromosome territory in precursor cells and more external in more differentiated cells. Our data suggest that such a repositioning of chromosomal loci during differentiation may be a consequence of general changes in chromosome territory morphology, not necessarily related to transcriptional changes. The radial distribution of chromosomes 1 and 8 also differed between cell types. In summary our data argue for a cell type specific chromosome territory organization in the investigated cell lines.cLys but not the #1 centromere and also other karyotype aberrations, e.g. a trisomy of chromosome 2. All lines were diploid for chromosome 8. Cells were cultured in IMDM supplemented with 2% chicken serum, 8% fetal calf serum and 2 mmol L-Glutamin and maintained at 37°C with 5% CO2. For 3D-FISH experiments cells were cultured on #1.5 glass coverslips (170 μm thick). Except for macrophages, coverslips were pretreated for 35 min with poly-L-lysine . Activation of HD11 was achieved by the addition of 5 μg LPS (Sigma, L-8275) per ml medium and subsequent cultivation over night [All cells are from retrovirally transformed chicken cell lines ,57. HD50er night  for 15 mChromosome-specific paint probes for chicken chromosomes 1 and 8 were kindly provided by Dr. Felix Habermann and Dr. Johannes Wienberg, Munich. They were generated by flow sorting of metaphase chromosomes and subsequent degenerated oligonucleotide primed (DOP)-PCR . They prcLys the following amounts of DNA were precipitated for each μl hybridization mix: 2 μl label-DOP-PCR product of an early amplification round of the #1 paint plus 2 μl of a highly amplified paint probe for centromere detection, 50 ng pPoly-III-i Lys, 2.5 μg cot 1 DNA. When paint probes for #1 and #8 were cohybridized, 2 μl label-DOP-PCR product of an early amplification round was used for each chromosome and supplemented with cot1 DNA as above. Denaturation was 5 min at 85°C. Preannealing with the cot 1 DNA was performed for 25 min at 37°C. For 3D FISH, coverslips with cells were denatured in 70% formamide for 3 min at 70°C, and placed immediately in ice-cold 50% formamide/2 × SSC. They were then incubated with 5 μl hybridization mix under a sealed 18 × 18 mm2 coverslip at 37°C for 24 h-72 h. Air-drying was carefully avoided at all steps. Metaphase chromosomes were hybridized as described [All DNA for a given assay was mixed, precipitated and solved in deionized formamide. The same volume of 20% dextransulfat in 2 × SSC was added. In experiments with Detection was performed as described , using r3D image stacks (8 bit) were recorded with a Leica TCS SP confocal laser scanning microscope equipped with an argon (488, 514 nm) and a HeNe laser (633 nm) (Leica Mikrosysteme, Bensheim, Germany). A 100 × N.A. 1,4 oil objective was used to obtain stacks with a voxel size of 0.08 × 0.08 × 0.24 μm. Nuclei with separated homologous chromosome territories were preferably selected for recording. To measure the chromatic aberration, 0.5 μm multi-color latex beads (Polysciences Europe, Eppelheim, Germany) were fed to activated macrophages. After phagocytosis the cells were fixed and embedded like 3D-FISH preparations. The beads were in the cytoplasma and thus their optical environment was closer to the situation of FISH signals than beads mounted directly on a glass cover slip. The chromatic aberration in x, y and z was corrected before the assignment of signals to categories or distance measurements were performed.The program used for object counting was first applied by Cremer et al. . The ori2. This value reflects the amount of intensity (chromatin) that is enclosed in a given surface area. Chromosome territories with a richly folded surface thus have a rather low value whereas compact, homogeneous territories have a higher value. Most nuclei had zero or few objects at very high threshold levels /(object surface voxels) was measured for all objects and averaged. Object surface voxels are defined as voxels belonging to an object and having at least one of the 26 neighbors not belonging to the object. The unit is 1/μms Figure . TherefocLys and centromere signals with regard to chromosome 1 territories: Image stacks were imported in ImageJ  and eacernet at . The MancLys and the respective centromere were corrected for the chromatic aberration and the distance was calculated.High precision 3D-distance measurements ,36: GravThe quantitative assessment of 3D radial distributions of painted chromosome territories and the measurement of nuclear volumes was performed using a program developed by Dr. Johann von Hase, Heidelberg which is described in detail elsewhere . To deteThe study was designed by SD together with CB and TC. First experiments and development of techniques were performed by RM. S Stadler and VS performed 3D-FISH, microscopy, determination of cLys and centromere 1 localization and high precision distance measurements of all evaluated cells. RM carried out radial distribution statistics and volume measurements. All three were supervised by SD and TC. S Stein programmed and adapted the object counting program, supervised by CC. Object counting and statistical analysis was performed by SD. The manuscript was written by SD with substantial contributions by CB, TC and VS. All authors read and approved the final manuscript.Radial distribution of chromosomes 1 and 8 in nuclei of different cell types. These graphs show the same curves as presented in Figure Click here for file


Some primatologists have argued that to understand human nature we must understand the behavior of apes. In the social interactions and organization of modern primates, the theory goes, we can see the evolutionary roots of our own social relationships. In the genomic era, the age-old question, What makes us human? has become, Why are we not apes? As scientists become more adept at extracting biological meaning from an ever expanding repository of sequenced genomes, it is likely that our next of kin will again hold promising clues to our own identity.Many comparative genomics studies have looked to our more distant evolutionary relatives, such as the mouse and even yeast, to help interpret the human genome. Because the genomes of mice, yeast, and humans have diverged significantly since their last common ancestor—about 75 million years ago for mouse and human, and about 1 billion years ago for yeast and human—there are enough differences between the functional and nonfunctional regions to home in on biologically significant sequences, based on their similarity. Sequences that are similar, or conserved, in such divergent species are assumed to encode important biological functions. These comparative studies have successfully identified and characterized many human genes. And a similar approach comparing primate genomes can help scientists understand the genetic basis of the physical and biochemical traits that distinguish primate species. In this approach, however, rather than looking for genes that are shared across many species, scientists look for those that are unique to a species.One of the primary agents of genome evolution is gene duplication. Duplicated genes provide the raw material for the generation of novel genes and biological functions, which in turn allow the evolution of organismal complexity and new species. (For more on duplicated genes, see the primer by Hurles in this issue.) James Sikela and colleagues set out to compare gene duplications between humans and four of our closest primate relatives to find the genetic roots of our evolutionary split from the other great apes. Collecting the DNA of humans, chimpanzees, bonobos, gorillas, and orangutans from blood and experimental cell lines, the researchers used microarray analysis to identify variations in the number of copies of individual genes among the different species. They analyzed nearly 30,000 human genes and compared their copy numbers in the genomes of humans and the four great apes.Overall, Sikela and colleagues found more than 1,000 genes with lineage-specific changes in copy number, representing 3.4% of the genes tested. All the great ape species showed more increases than decreases in gene copy numbers, but relative to the evolutionary age of each lineage, humans showed the highest number of genes with increased copy numbers, at 134. Many of these duplicated human genes are implicated in brain structure and function.The gene changes identified in the study, the authors conclude, likely represent most of the major lineage-specific gene expansions (or losses) that have taken place since orangutans split from the other great apes, some 15 million years ago. (Humans diverged from their closest cousins, the chimp and bonobo, roughly 5 million to 7 million years ago.) And because some of these gene changes were unique to each of the species examined, they will likely account for some of the physiological and morphological characteristics that are unique to each species. One cluster of genes that amplified only in humans was mapped to a genomic area that appears prone to instability in human, chimp, bonobo, and gorilla. This region, which corresponds to an ancestral region in the orangutan genome, has undergone modifications in each of the other descendent primate species, suggesting an evolutionary role. In humans, gene mutations in this region are also associated with the inherited disorder spinal muscular atrophy. This fact, along with the observation that there are human-specific gene duplications in this region, suggests a link between genome instability, disease processes, and evolutionary adaptation.In their genome-wide hunt for gene duplications and losses in humans and great apes, Sikela and colleagues have highlighted genomic regions likely to have influenced primate evolution. With the impending release of the chimp genome and more primate sequences to follow, scientists can take advantage of both sequence-based and microarray-based genome information to wrest additional insights from our primate cousins and flesh out the details of the human story.


Ultrasound-mediated microbubbles destruction has been proposed as an innovative method for noninvasive delivering of drugs and genes to different tissues. Microbubbles are used to carry a drug or gene until a specific area of interest is reached, and then ultrasound is used to burst the microbubbles, causing site-specific delivery of the bioactive materials. Furthermore, the ability of albumin-coated microbubbles to adhere to vascular regions with glycocalix damage or endothelial dysfunction is another possible mechanism to deliver drugs even in the absence of ultrasound. This review focuses on the characteristics of microbubbles that give them therapeutic properties and some important aspects of ultrasound parameters that are known to influence microbubble-mediated drug delivery. In addition, current studies involving this novel therapeutical application of microbubbles will be discussed. The recent advances in gene therapy and molecular biology have improved the interest in methods of noninvasive delivery of therapeutic agents. Besides the well known application of microbubbles as contrast agents for diagnostic ultrasound, microbubbles have also been demonstrated an effective technique for targeted delivery of drugs and genes -6. DrugsTwo possible strategies for delivering drugs and genes with microbubbles are emerging. The first consists on the ultrasound-mediated microbubble destruction, which is based on the cavitation of microbubbles induced by ultrasound application, and the second is the direct delivery of substances bound to microbubbles in the absence of ultrasound. Different drugs and genes can be incorporated into the ultrasound contrast agents. It has already been demonstrated that perfluorocarbon-filled albumin microbubbles avidly bind proteins and synthetic oligonucleotides . In a siThe mechanisms by which ultrasound facilitates the delivery of drugs and genes result from a complex interplay among the therapeutic agent, the microbubble characteristics, the target tissue, and the nature of ultrasound energy. The presence of microbubbles in the insonified field reduces the peak negative pressure needed to enhance drug delivery with ultrasound. This occurs because the microbubbles act as nuclei for cavitation, decreasing the threshold of ultrasound energy necessary to cause this phenomenon. The results of optical and acoustical studies have suggested the following mechanisms for microbubble destruction by ultrasound: 1- gradual diffusion of gas at low acoustic power, 2- formation of a shell defect with diffusion of gas, 3- immediate expulsion of the microbubble shell at high acoustic power, and 4- dispersion of the microbubble into several smaller bubbles. Cavitation of the bubbles is characterized by rapid destruction of contrast agents due to a hydrodynamic instability excited during large amplitude oscillations, and is directly dependent on the transmission pressure ,18. It h2. However, it was a lower intensity of ultrasound (0.6 W/cm2) that caused more drug delivery with microbubbles. More recently, voltage clamp techniques were used to obtain real-time measurements of membrane sonoporation in the presence of albumin-coated microbubbles (Optison). Ultrasound increased the transmembrane current as a direct result of membrane resistance due to pore formation [The formation of pores in the membranes of cells as a result of ultrasound-induced microbubble cavitation has been proposed as a mechanism for facilitating the drug deposition. Taniyama et al  demonstrormation .Another important therapeutic property of microbubbles is their increased adherence to damaged vascular endothelium. Albumin-coated microbubbles do not adhere to normally functioning endothelium, but their adherence does occur to activated endothelial cells or to extra-cellular matrix of the disrupted vascular wall, and this interaction could be a marker of endothelial integrity . BecauseThe clinical use of viral vectors for gene therapy is limited because viral proteins elicit an immune response within the target tissue , and havTaniyama et al  have alsmyc protooncogene is responsible for the regulation of gene expression involved in the process of intimal hyperplasia that leads to restenosis. Synthetic antisense oligonucleotides, such as those to the c-myc protooncogene, can bind to the messenger ribonucleic acid and inhibit the synthesis of the protooncogenes. Therefore, antisense to c-myc protooncogene can prevent its translation into proteins that may be mediators of the pathologic process of restenosis. These synthetic agents, when administered directly into the vessel, have successfully inhibited restenosis after coronary or carotid injury [myc, 21 pigs had carotid balloon injury performed with an oversized balloon catheter and were randomized to receive intravenous antisense to c-myc bound to PESDA, intravenous antisense alone, or no treatment. The pigs that received antisense bound to PESDA also had transcutaneous 20 kHz ultrasound applied over the carotid wall following injections. The ultrasound targeted group showed a significantly lower percent area stenosis (8 ± 2%) than the two control groups  [Restenosis after vascular balloon injury or stent deployment has been shown to result from neointimal hyperplasia due to smooth muscle cell migration and proliferation. The c-d injury . In 1996d injury  demonstrmyc into coronary arteries using high phase liquid chromatography in pigs. Intravenous PESDA containing anti c-myc was given in the presence or absence of transthoracic 1 MHz ultrasound (0.6 W/cm2). In this study, the authors demonstrated that anti c-myc can be selectively concentrated within a stretch-injured coronary artery segment when given intravenously bound to PESDA. The decrease in neointimal formation following intravenous injection of anti c-myc with PESDA without ultrasound was similar to that observed with higher doses of the same antisense given directly into the coronary artery using an infiltrator delivery system [Since PESDA microbubbles adhere to sites of endothelial injury even in the absence of ultrasound, the efficacy of this therapy in inhibiting coronary restenosis has been evaluated in animals. Porter et al  measuredy system . The basy system ,21,31. Ay system . Since ly system , it is py system , and thuy system . Figure y system  have alsy system .Another innovative application of microbubbles and ultrasound is in the delivery of proteins that induce growth of endothelial cells, such as vascular endothelial growth factor (VEGF). Mukherjee et al  demonstrmyc protooncogene (0.19 ± 0.04 μg/mg), but low-frequency 20 kHz ultrasound significantly increased vascular uptake  when compared to antisense bound to PESDA alone (0.21 ± 0.06 μg/mg). The results of this study suggested that a lower frequency could be better suited to target antisense deposition into major vessels. Because there were minimal differences in peak negative pressure generated by 2 MHz and 20 kHz in this study (46 kPa and 13 kPa, respectively), the enhanced uptake was attributed to a lower threshold for cavitation with 20 kHz ultrasound frequency.The effect of several ultrasound parameters, including transducer frequency and acoustic power, are known to influence microbubble destruction and, thus, the transfection of genes and drugs. Although the optimal ultrasound parameters for maximizing this process are not known, we will briefly discuss some important aspects. Unger et al  have sho2). The authors found a significant increase in VEGF uptake with the combination of ultrasound and PESDA at all power outputs when compared with controls, but there was a dose-dependent increase in the amount of VEGF uptake with increasing power until 0.6 W/cm2 and a subsequent plateau. Table In another study, the efficacy of ultrasound-mediated delivery of VEGF bound to PESDA into the myocardium of rats was evaluated with an ultrasound frequency of 1.0 MHz at various acoustical outputs (0.2, 0.4, 0.6, 0.8 and 1.0 W/cmHowever, a high peak negative pressure may have detrimental bioeffects. Several investigators have reported on the occurrence of tissue hemorrhage and endothelial cell damage after ultrasound exposure of cultured cells and organs containing air, such as the lungs or the intestine -39. Ay eDr. Jeane M. Tsutsui – declares no competing interests.Dr. Feng Xie – declares no competing interests.Dr. Thomas R. Porter – declares ImaRx Therapeutics, Inc.: Grant support and Consultant; Bristol Myers Squibb Medical Imaging: Grant support; AVI BioPharma, Inc.: Grant support


Early treatment of acute HIV infection with highly active antiretroviral therapy, followed by supervised treatment interruption (STI), has been associated with at least transient control of viremia. However, the durability of such control remains unclear. Here we present longitudinal follow-up of a single-arm, open-label study assessing the impact of STI in the setting of acute HIV-1 infection.Fourteen patients were treated during acute HIV-1 infection and subsequently subjected to an STI protocol that required retreatment if viral load exceeded 50,000 RNA copies/ml plasma or remained above 5,000 copies/ml for more than three consecutive weeks. Eleven of 14 (79%) patients were able to achieve viral loads of less than 5,000 RNA copies/ml for at least 90 d following one, two, or three interruptions of treatment. However, a gradual increase in viremia and decline in CD4+ T cell counts was observed in most individuals. By an intention-to-treat analysis, eight (57%), six (43%), and three (21%) of 14 patients achieved a maximal period of control of 180, 360, and 720 d, respectively, despite augmentation of HIV-specific CD4+ and CD8+ T cell responses. The magnitude of HIV-1-specific cellular immune responses before treatment interruption did not predict duration of viremia control. The small sample size and lack of concurrent untreated controls preclude assessment of possible clinical benefit despite failure to control viremia by study criteria.These data indicate that despite initial control of viremia, durable viral control to less than 5,000 RNA copies/ml plasma in patients following treated acute HIV-1 infection occurs infrequently. Determination of whether early treatment leads to overall clinical benefit will require a larger and randomized clinical trial. These data may be relevant to current efforts to develop an HIV-1 vaccine designed to retard disease progression rather than prevent infection since they indicate that durable maintenance of low-level viremia may be difficult to achieve. Long-term follow-up of 14 patients on HAART during acute infection followed by supervised treatment interruptions shows that most failed to achieve lasting control of viremia. The use of highly active antiretroviral therapy (HAART) can dramatically prolong the life of individuals infected by human immunodeficiency virus 1 (HIV-1) . The sucIn the present study, we performed a detailed longitudinal assessment of the impact of early treatment followed by STIs in patients treated during acute or early HIV-1 infection. The main hypothesis of the study was that early treatment of acute HIV-1 infection followed by STI would lead to immune boosting and subsequent control of viremia without the need for drugs. The primary endpoint was the time to viral rebound above 50,000 copies/ml once or above 5,000 copies for three determinations separated by a week each. The early results of this trial were previously reported, showing that five of eight patients were able to achieve a plasma viral load of 500 copies/ml or less at a median of 6 mo off therapy [The hypothesis of the study was that early treatment of acute HIV-1 infection would confer immunologic maturation and subsequent control of HIV-1 without the need for ongoing drug therapy. Alternatively, if a breakthrough of virus replication was observed, this would provide a boost in HIV-1-specific immunity after reinstitution of antiviral therapy. The primary endpoint was the time to viral rebound to more than 50,000 copies/ml or viral loads above 5,000 copies/ml for three determinations separated by a week each. The secondary objective was to correlate immunologic and virologic parameters with any observed effects including evolution of HIV-1-specific T helper and cytotoxic T lymphocyte responses. The original study protocol, including the patient consent form and the institutional review board approval, can be found in Fourteen patients presenting with acute or early HIV-1 infection were enrolled in this study between July 1997 and January 2000 . Acute HSix of the 14 individuals were investigated in an interim study . These pEntry criteria included treatment with HAART before or shortly after HIV-1 seroconversion, viral suppression on HAART to less than 400 RNA copies/ml for 2 mo, HIV-1 viral load at the time of entry into the study of less than 50 RNA copies/ml, and lack of significant mutations conferring drug resistance ,12. LympHigh- and intermediate-resolution human leukocyte antigen class I typing was performed at a commercial laboratory  by sequence-specific PCR as described .GB virus C (GBV-C) RNA was detected using a two-step nested PCR amplification reaction from whole plasma RNA. Briefly, GBV-C RNA was extracted from plasma using the Qiagen Viral RNA Mini Kit  according to the manufacturer's instructions. Extracted RNA was reverse transcribed using the Qiagen OneStep RT-PCR Kit and amplified by nested PCR; in both steps primers specific for the 5′ UTR of GBV-C were used, as described previously .In order to analyze the chemokine receptor (CCR) 5Δ32 deletion polymorphism, the region spanning the 32-nt deletion was amplified by PCR, and the two alleles were separated by gel electrophoresis . The CCRWe synthesized 410 synthetic peptides 15–20 amino acids long at the Massachusetts General Hospital Peptide Core Facility on an automated peptide synthesizer using Fmoc technology, as described . Peptide6 PBMCs and if the well had at least three times the mean number of SFCs in the three control wells. The dependence of responses on CD8+ T cells was determined by measuring the depletion of CD4+ T cells using the Minimacs cell depletion system (Miltenyi Biotec, Bergisch-Gladbach, Germany). When HIV-1-specific CD8+ T cell responses were detected against adjacent peptides, and therefore might represent targeting of the overlap region, responses to the weaker peptide were excluded for calculations of magnitude and breadth, as previously described [ELISPOT assays were carried out as described previously . Peripheescribed .5 cells) were incubated with baculovirus-derived recombinant p24 protein (Protein Sciences, Meriden, Connecticut, United States) at 5 μg/ml for 6 d and then pulsed with 3H thymidine at 1.0 μCi for 6 h before harvesting as previously described [Freshly isolated PBMCs  were able to achieve virologic control to less than 5,000 RNA copies/ml plasma for at least 90 d after one, two, or three treatment interruptions . The perOnce control was achieved, the majority of the patients experienced a subsequent rise in viremia. The median time between cessation of therapy and rebound of viremia  was 17 d (range, 7–169 d).Six of 14 patients (43%) achieved a period of control after stopping therapy for 1 y, but only three of 14 (21%) were able to control viremia off therapy at less than 5,000 RNA copies/ml plasma for more than 2 y. Duration of viremia control during successive treatment interruptions was highly variable, and there was no increase in the sustainability of viral containment during successive STI cycles. The three patients achieving control of viremia for more than 2 y did so during the first (AC-10), the second (AC-02), and the third (AC-14) treatment interruption, respectively A. A pairThese data show that at least transient control of viremia to less than 5,000 RNA copies/ml plasma was achieved in the majority of study participants during at least one of the treatment interruptions, but that durable viral control in participants following treated acute infection occurred infrequently. Moreover, the data do not show a consistent pattern of augmentation of viral control with sequential treatment interruptions.3; range, 492–986), but the effect of treatment interruption was variable, even in those doing well, as defined by sustained low viral loads. Overall, 11 of 14 patients interrupted therapy for at least 12 mo, and these individuals were evaluated regarding the effect of treatment interruption on CD4+ T cell loss (3/y (median, −192). Of the three patients who did not meet viral load criteria for restarting therapy for more than 2 y, one (AC-02) had an increasing CD4+ T cell count of 157 cells/mm3/y, one (AC-10) had a stable CD4+ T cell count (−9 cells/mm3/y) , and one (AC-14) experienced a decline of 344 cells/mm3/y. Comparison with data from the Multicenter AIDS Cohort Study (MACS) showed that the kinetics of CD4+ T cell loss was faster (Mann-Whitney U test , p = 0.02) than in untreated patients with early chronic HIV-1 infection (average loss of −67 cells/mm3/y in patients with a CD4+ T cell count of more than 350 cells/mm3 at baseline). However, CD4+ T cell loss rate was in the same range as what has been described after treatment interruption in chronic HIV-1 infection [3/y; AC-10, +44 cells/mm3/y; and AC-14, −110 cells/mm3/y. When the first 3 mo off therapy were excluded in order to minimize the potential effects of recent treatment on CD4+ T cell number, the rate of change in CD4+ T cell counts during the first year off therapy no longer differed statistically from the MACS data . A possible reason for steep CD4+ T cell slopes may be high CD4+ T cell counts at time of treatment interruption. Comparison with MACS data . The individuals who controlled viremia for a longer time either during the first STI or during any of the treatment interruptions were not different from those who experienced earlier breakthrough as measured by ELISA and Western blot status at initiation of HAART, coreceptor polymorphisms (CCR5delta32, CCR2 V64I), or the presence or absence of GBV-C coinfection . The only parameter that was predictive of prolonged viral control during the first treatment interruption was a low viremia at time of institution of therapy (p = 0.01): there was a 2.8-fold increase in hazard per order of magnitude increase in viral load. This factor was no longer predictive when the period of longest control of viremia was considered. The time to rebound of viremia (>50 copies/ml or >400 copies/ml) did not correlate with the duration of viral control. Although 11 out of 14 individuals achieved at least transient control of viremia, and three experienced prolonged control, none of these patients possessed the HLA alleles B27 or B57 associated with better disease outcome [Although the study was small, we evaluated clinical and laboratory parameters to see if any was predictive of duration of viral control. Analyses included clinical and laboratory parameters at time of presentation with acute HIV-1 infection, genetic markers associated with different rates of disease progression, and the presence or absence of GBV-C coinfection. All patients presented with symptomatic acute infection. Time between onset of symptoms and institution of therapy did not affect duration of control following STI  (p < 0.05) during the first off-treatment period, reaching a median total magnitude of 2,725 SFCs/106 PBMCs and targeting a median of eight epitopes, and then were sustained when therapy was reintroduced. A further increase in the magnitude and breadth of HIV-1-specific CD8+ T cells was observed in the subsequent off-treatment periods, although these augmentations failed to reach statistical significance. The CD8+ T cell–mediated immune responses emerging during these consecutive cycles of treatment interruption were broadly directed, targeting all structural and most accessory and regulatory HIV-1 gene products (data not shown). However, the magnitude of HIV-1-specific CD8+ T cell responses at the beginning of the first (r = 0.01, p = 0.76), second (r = 0.16, p = 0.54), or third (r = 0.1, p = 0.55) treatment interruptions was not predictive of the time the study participants were subsequently able to stay off therapy according to study criteria.To assess the relationship between the clinical outcome and evolution of HIV-1-specific CD8+ T cells, we longitudinally analyzed the breadth and magnitude of CD8+ T cell responses using an interferon-γ ELISPOT and a panel of 410 overlapping peptides spanning the entire HIV-1 clade B consensus sequence. At the beginning of the first STI, HIV-1-specific CD8+ T cells were weak (median of 590 SFCs/106 PBMCs) A and nar6 PBMCs) B. CD8+ Tr = 0.92, p < 0.001) . The increase in CD8+ T cell epitopes targeted during the first treatment interruption was also linearly correlated to the duration until virological failure (r = 0.81, p < 0.001)  C. Simila< 0.001) D. Howevep = 0.72; second, p = 0.12; and third, p = 0.60, respectively). These HIV-1-specific CD4+ T cell responses also tended to rise with reinitiation of therapy, and some of them were very robust, with stimulation indices over 50 detected in several individuals (r = 0.05, p = 0.43) (r = 0.16, p = 0.54), or third (r = 0.1, p = 0.55) treatment interruption was not statistically predictive of the time the study participants were subsequently able to stay off therapy according to study criteria.We next analyzed evolution of lymphoproliferative responses to recombinant HIV-1 p24 Gag protein in order to assess HIV-1-specific CD4+ T cell function. Most individuals had no detectable response at baseline prior to treatment, consistent with prior reports of patients with acute HIV-1 infection . After iividuals E. Simila = 0.43) F, secondAlthough early treatment of acute HIV-1 infection followed by treatment interruptions may enhance control of viremia ,8, the dThe standard immunologic assays and virologic assessments in this cohort revealed considerable heterogeneity among the study participants, and did not show a consistent pattern in duration of viremia control during successive treatment interruptions. Eleven of 14 patients (79%) were able to maintain a viral load of less than 5,000 copies/ml for at least 90 d, but progressive loss of control ensued in the majority of patients and only three patients (21%) were able to maintain control for more than 2 y. These three patients did so during the first (AC-10), the second (AC-02), and the third (AC-14) STI. Clinical, genetic, and immunological parameters did not distinguish these three individuals from the other 11 patients, nor did they predict the duration of control following treatment interruption. Indeed, the longer a patient was off therapy, the stronger and more broadly directed the CD8+ T cell responses became, but these were still not sufficient to maintain prolonged control in most patients. Although three patients did not complete the study as initially intended , the results are not substantially different if these three are censored rather than considered to have failed to control.Loss of viral control in this cohort occurred not only in the presence of strong CD8+ T cell responses, but in most cases also in the presence of virus-specific CD4+ T cell responses, although the CD4+ T cell responses often declined during periods of viremia. In addition, total CD4+ T cell numbers were also monitored and declined in most patients over time, including one of the three patients who were able to maintain low viral loads for at least 2 y. Mechanisms leading to rapid CD4+ T cell loss need to be further studied in future STI trials. Other parameters including chemokine receptor polymorphisms  and GBV-The reasons for progressive loss of control despite augmentation of virus-specific CD4+ and CD8+ T cell responses remain to be defined. In one individual (AC-06), HIV-1 superinfection in the setting of strong and broadly directed HIV-specific cellular immune responses was associated with the loss of viral control, as previously reported . No otheThese data are important in light of other recent data on treatment interruption in both acute and chronic infection. In chronic HIV-1 infection, STI studies showed only marginal, if any, improvements of HIV-1 viremia control following a number of treatment interruptions cycles, despite at least transient increases in HIV-1-specific CD8+ and CD4+ T cell responses ,53,54,55Although durable control of viremia was not achieved, it is noteworthy that the majority of patients were able to achieve transient relative containment of viremia, providing rationale for future studies aimed at further enhancing immune control. Early treatment alone should still be considered an important therapeutic option. Therapeutic vaccinations administered after treated acute HIV-1 infection and before cessation of therapy have given disappointing results thus far , but theAlthough the present study shows progressive viral breakthrough, it was not designed to address whether there might be a change in set point viremia achieved or overall clinical benefit through transient early treatment of acute HIV infection. The definition of failure chosen for this study was a viral load of greater than 5,000 RNA copies/ml plasma, which at the time the study was initiated corresponded to the level of viremia at which treatment was recommended. Larger randomized trials will be needed to determine the potential clinical and virologic benefit of approaches based on STIs. In studies of untreated infection, there is only a 5-fold difference in viremia separating the quartile with the slowest disease progression from the quartile with the most rapid progression , suggestProtocol S1(68 KB DOC).Click here for additional data file.Protocol S2(47 KB PDF).Click here for additional data file.Protocol S3(187 KB PDF).Click here for additional data file.Protocol S4(36 KB PDF).Click here for additional data file.Highly active antiretroviral therapy (HAART), which is used to treat patients with HIV, can have nasty side effects and is expensive. As a result, for the last five years scientists have been trying to determine if it is possible to give patients breaks from taking antiretroviral drugs, without patients' health suffering. It is likely that such a treatment strategy  will not work in patients who have been infected with HIV for a long time. However, until now, the jury was out about whether STI could be of benefit to patients who had only recently been infected with HIV. Some research suggested that if newly infected patients had short “holidays” from taking HAART, it might help to boost their immune system—which in turn might help to keep HIV at bay.The researchers studied 14 patients who had recently been infected with HIV. Patients were treated until their viral load was below the limit of detection by using a very sensitive method. As they were then watched after treatment interruption, most of them were able to maintain a low, though detectable, viral load for some time. The researchers then stopped the treatment and carefully watched the patients over a period of up to five years. If the number of viruses in the patients' blood rose too high, the researchers gave the patients HAART again until the number of viruses fell again. In about half the patients, treatment needed to be restarted within a year because the viruses had started becoming more numerous in the blood. The doctors had hoped that it would take much longer for the number of viruses to reach this level. In other words, giving patients “drug holidays” did not help to keep the virus at bay for long periods of time.It is relatively rare for doctors to be able to diagnose HIV shortly after an individual becomes infected with the virus. Many people have HIV for a long time before they see a doctor. Therefore, even if the STI strategy had worked, it would only have been relevant to a few patients. In addition, the study was small and preliminary, so we have to be careful about reading too much into the results. A limitation in the study is that it did not address whether there might be a long-term clinical benefit despite a gradual increase in viral load.It is important to remember that this was an experimental trial—patients with HIV should not stop taking antiretroviral drugs unless their doctor specifically tells them to do so. Although it looks as though STI may not work in the way everyone had hoped, these results may help scientists develop an HIV vaccine that is designed to keep the disease at bay. Whether early treatment of acute infection has an overall benefit in terms of time-to-development of AIDS or need for long-term treatment after drug discontinuation will need to be answered in larger clinical trials designed to answer these important questions.http://www.aidsinfo.nih.gov/AIDSinfo: http://www.aidsmap.com/AIDSmap: http://www.nlm.nih.gov/medlineplus/aids.htmlMedline Plus AIDS Information:


The Microarray Gene Expression Data Society believe that the time is right for journals to require that microarray data be deposited in public repositories, as a condition for publication While some journals have already made this a condition of acceptance, we feel that submission requirements should be applied consistently and that journals should recognize ArrayExpress , European Bioinformatics Institute (EBI), and National Center for Biotechnology Information (NCBI). Similarly, we, members of the Microarray Gene Expression Data Society  as a means of achieving this goal. (2) Scientific journals should require that all primary microarray data are submitted to one of the public repositories—ArrayExpress, GEO, or CIBEX—in a format that complies with the MIAME guidelines. (3) Public databases should work with authors and scientific journals to establish data submission and release protocols to assure compliance with MIAME guidelines. (4) To assist with the review process, the databases should continue to work in collaboration with publishers to provide qualified referees with secure means of accessing prepublication data. Authors should be strongly encouraged to submit data to the databases during review.To this end, the members of MGED propose the following as a new paradigm for the publication of microarray-based studies. (1) Authors should continue to take primary responsibility for ensuring that all data collected and analyzed in their experiments adhere to the “Minimum Information about a Microarray Experiment” (MIAME) guidelines and should continue to use the MIAME checklist , ArrayExpress (www.ebi.ac.uk/arrayexpress), and CIBEX (cibex.nig.ac.jp), as well as a growing number of commercial and academic software packages capable of writing MAGE-ML documents  These established repositories have a commitment to continued community service and to providing some level of assurance that published gene expression datasets will continue to be available into the future. (2) Having the data available in these public repositories in a standardized format will not only make them more accessible, but it will allow expression data to be integrated with other relevant data, including the available genome sequences, single nucleotide polymorphism and haplotype mapping information, the literature, and other resources that can aid in further interpretation of expression patterns. Although many authors now provide some or all of this information, the established databases are much more likely to assure that these links are maintained and current. (3) Curation of data submitted to public data repositories will assist authors, reviewers, and publishers in assuring that the data comply with the MIAME requirements, further enhancing their utility. (4) The standardization of microarray data formats will enable the development of additional data analysis and integration tools and makes it easier for scientists to access, query, and share data. (5) Finally, submission prior to publication will make it easier for referees to access the data confidentially, facilitating the review and publication process.In the same way that availability of sequence data had a profound impact on a wide range of disciplines, we believe that requiring that microarray data be deposited in public repositories as a necessity for publication will accelerate the rate of scientific discovery.What this proposal requires is a change in the way in which we approach the publication of microarray-based studies. Both authors and journals have a responsibility to assure that the requisite data are available, and because submitting MIAME-compliant data can take considerable time and effort, this process should be factored into review and publication timelines. However, while this process may be time consuming and painful at first, we believe that the benefits of building an open repository of microarray data will far outweigh any initial disadvantages. As always, it is our sincere hope that these suggestions stimulate discussion within the community and that together we can arrive at a consensus that ensures that microarray data are widely and easily accessible. Finally we would like to urge the DDBJ, EBI, and NCBI to work together towards exchanging all MIAME-compliant microarray data.


Tumors and complex tissues consist of mixtures of communicating cells that differ significantly in their gene expression status. In order to understand how different cell types influence one another's gene expression, it will be necessary to monitor the mRNA profiles of each cell type independently and to dissect the mechanisms that regulate their gene expression outcomes.In order to approach these questions, we have used RNA-binding proteins such as ELAV/Hu, poly (A) binding protein (PABP) and cap-binding protein (eIF-4E) as reporters of gene expression. Here we demonstrate that the epitope-tagged RNA binding protein, PABP, expressed separately in tumor cells and endothelial cells can be used to discriminate their respective mRNA targets from mixtures of these cells without significant mRNA reassortment or exchange. Moreover, using this approach we identify a set of endothelial genes that respond to the presence of co-cultured breast tumor cells.RNA-binding proteins can be used as reporters to elucidate components of operational mRNA networks and operons involved in regulating cell-type specific gene expression in tissues and tumors. Many recent studies have described the use of microarrays to identify genes expressed in different types of cancers . Our laIn this study, we define a model system for using poly (A) binding protein (PABP) to recover mRNAs from specific cell-types in mixed cell cultures. Using this approach, we were able to determine how the gene expression profiles of endothelial cells change in response to the presence of breast cancer cells. Among the advantages of this approach are: a) no manipulations or treatments are required prior to the preparation of cell extracts, b) the recovered mRNA population can be identified directly using genomic methods, and c) RNA binding proteins can be engineered for expression in different cell types using various molecular tags in order to discriminate cell-specific mRNA populations. These studies provide a methodological basis for creating mouse models in which different types of cells within a tumor express RNA binding proteins to reveal unique populations of posttranscriptionally regulated mRNAs.The goals of these experiments are to validate procedures for the isolation and characterization of discrete mRNA sub-populations associated with RNA binding proteins expressed in specific cell types within a tumor or organ in order to assess the responses of cells to their surroundings. Earlier studies have shown that mRNA subpopulations in single cell types reflect the functions of the RNA binding proteins with which they associate and can provide key information about post-transcriptional regulatory mechanisms of gene expression ,13,15-18Microarray analysis was used to compare the gene expression profiles obtained using total RNA and PABP-associated RNA of PY 4.1 endothelial cells  with proteins (proteome) in the same cell population ,29. The It is possible that gene expression profiles obtained by immunoprecipitating mRNA-PABP complexes may reflect the functional state of protein production from these mRNAs . For theIn order to compare mRNA profiles from mixed cell populations, we prepared two different cell lines stably expressing different epitope tagged PABPs. Figure A potential complication for this type of analysis is that expression of a tagged-RNA binding protein, in this case PABP, could affect cell growth. While these cell lines appeared unaffected morphologically, the levels of PABP in cell lines expressing tagged-PABP and respective control cell lines were evaluated and compared by Western blotting. No substantial change in overall PABP expression was observed when expressing exogenous tagged PABP Figure . This reNo changes in cell growth or mortality of the cell lines used in this study or in other cells lines expressing tagged-PABP were observed. Moreover, the cell cycle kinetics of T98G cells expressing Flag-PABP and cells that were subsequently stimulated by serum addition were compared to those of T98G cells containing the empty vector, pCMVneo and no substantial differences were observed using fluorescent cell sorting Figure . We concEarly studies of PABP binding to mRNA indicated that a dynamic exchange or hopping of PABP from mRNA to mRNA might be an important aspect of its function ,34. For We have also addressed the potential problem of having a pool of free PABP in an extract that could be available to bind mRNA during incubation. Sucrose gradient analysis indicated that this is very unlikely since the majority of PABP was found in heavy polysomes and associated with mRNA, while only a small percentage was found in the upper portion of the sucrose gradients (H.S. and J.D.K., unpublished data). In total, these results demonstrate that reassortment of PABP in these cell extracts was not a significant limitation to using PABP RNPs for gene expression profiling of bound mRNAs.As noted above, it has been suggested that yeast PABP uses a "hopping" mechanism in vivo by moving from RNA to RNA ,34. Whilmouse angiogenesis (mAngio) and human tumor suppressor (hTS1) RPA probe sets after they were tested for cross species hybridization. Both probe sets showed good specificity of discrimination with the exception of the L32 and GAPDH control genes as expected . The mouse RNA on human Atlas arrays did not show any detectable cross hybridization signal, while the human RNA on mouse Atlas arrays showed a small percent (2–3%) of cross hybridizing mRNAs  were enriched at least 4 fold in the G10 PABP population when compared to the Flag PABP population. For the human genes, 82.4 % (122 out if 148) were enriched at least 4 fold in the Flag PABP population in relation to the G10 PABP population  often precede downstream alterations in the expression of other important genes. Among the RNA binding proteins identified in our screen were stem-loop binding protein, a highly conserved RNA binding protein that binds a stem loop structure in the 3'UTR of histone mRNAs and is required for both processing and translation of histone messages [Drosophila bruno, a translation repressor which functions at the early steps of embryogenesis [As expected, the great majority of the genes expressed in PY4.1 cells were not altered in their levels of expression while in co-culture. Of interest, a small number of genes were consistently upregulated in four independent experiments. To assess the consistency of the fold change, we plotted the p-value (from modified t-test) against the average fold change Figure . A 'volcmessages , Brul4, ogenesis , and quaogenesis . It shouThe presence of several RNA binding proteins among the top-20 genes affected by co-cultivation may result in downstream effects on gene expression, and we plan to examine the target mRNAs of these RNA binding proteins in endothelial cells. This should help elucidate additional post-transcriptional pathways and networks regulating cell growth mechanisms and tumorigenesis .This study describes changes in the gene expression profile of an endothelial cell when co-cultivated with a tumor cell by isolating ribonucleoprotein complexes and identifying their associated mRNAs using genomic arrays. Moreover, it presents a model system that can be used to elucidate post-transcriptional operons in specific types of cells by using various RNA binding proteins from mixed cell cultures as a novel approach to understanding how cell-cell communication affects gene expression during tumorigenesis and organogenesis.Murine endothelial PY4.1 cells were kindly provided by Dr. Christopher Kontos, Duke University Medical Center. Human gliobastoma T98G and murine breast cancer 4T1 cells were obtained from American Type Culture Collection. All cell lines were maintained in DMEM Medium (Gibco) supplemented with 10% Fetal Bovine serum.The ORF of human PABP I containing the Flag tag (GACTACAAGGACGACGATGACAAG) or the G10 tag (CCACCATGGCT AGCATGACTGGTGGACAGCAAATGGGT) at the 5' end was cloned into the pCMV-Neo retroviral vector . Stable Monoclonal anti-G10 antibodies were obtained as previously described . AntibodProtein extracts were prepared from T98G and PY4.1 cell lines by homogenization in polysomal lysis buffer . 50 μg oAnalysis of the cell cycle of T98G cells was performed as described .Cell lysates and immunoprecipitation of mRNP complexes were essentially performed as described . PolyadeTotal or immunoprecipitated RNAs were assayed by RNase protection by using the PharMingen Riboquant assay according to the manufacturer's recommendations (45014K). mAngio-1 (mouse angiogenesis) and hTS1 (human tumor suppressor) template sets were used (551418 and 556161, respectively). Protected riboprobe fragments were visualized on a phosphorimaging screen (Molecular Dynamics). Phosphorimages were scanned by using the Molecular Dynamics STORM 860SYSTEM at 100 μm resolution and analyzed by using Molecular Dynamics IMAGE QUANT software (version 5.0).32P α-dATP (Amersham Biosciences). After hybridization, the array membrane was washed and the results were visualized on a phosphorimaging screen (Molecular Dynamics). Phosphorimages were scanned by using the Molecular Dynamics STORM 860SYSTEM at 100 μm resolution and stored as .gel files. Images were analyzed by using ATLASIMAGE 2.01 software (CLONTECH). Global normalization was used when arrays being compared had approximately the same number of positive hits.cDNA array analysis was performed by using Atlas Mouse and Human 1.2 Arrays (CLONTECH). Probing of cDNA arrays was performed as described in the CLONTECH Atlas cDNA Expression Arrays User Manual (PT3140-1). Reverse-transcribed probes were radiolabeled with .Printed oligo arrays using the Operon Mouse Oligo set version 2.0 (16,423 genes) were produced by the Duke Microarray Core Facility. Protocols used for preparation of slides, labeling, amplification, hybridization and scanning are described in GenePix data were normalized with pin-tip specific lowess normalization . DiffereELAV – embryonic lethal abnormal visionRPA – RNase Protection AssayPABP – Poly A binding proteinLOFP was responsible for the experimental design and performed Western blots, immunoprecipitations, RPAs and microarray experiments. MDB helped perform RPAs and microarray experiments. SML performed statistic analysis of microarray data and prepared the webpage with supplementary data. HS generated the stable cell lines used in this study and performed the cell cycle experiment. JDK conceived the project and assisted in experimental design.