Preservation of skin DNA for oligonucleotide array CGH studies: a feasibility study

Array-based comparative genomic hybridization (a-CGH) is a promising tool for clinical genomic studies. However, pre-analytical sample preparation methods have not been fully evaluated for this purpose. Parallel sections of normal male human skin biopsy samples were collected and immediately immersed in saline, formalin and a molecular fixative for 8, 12 and 24 h. Genomic DNA was isolated from the samples and subjected to amplification and labeling. Labeled samples were then co-hybridized with normal reference female DNA to Agilent oligonucleotide-based a-CGH 44k slides. Pre-analytic parameters such as DNA yield, quality of genomic DNA and labeling efficacy were evaluated. Also microarray analytical variables, including the feature signal intensity, data distribution dynamic range, signal to noise ratio and background intensity levels were assessed for data quality. DNA yield and quality of genomic DNA—as evaluated by spectrophotometry and gel electrophoresis—were similar for fresh and molecular fixative-exposed samples. In addition, labeling efficacy of dye incorporation was not drastically different. There was no difference between fresh and molecular fixative material comparing scan parameters and stem plot analysis of a-CGH result. Formalin-fixed samples, on the other hand, showed various errors such as oversaturation, non-uniformity in replicates, and decreased signal to noise ratio. Overall, the a-CGH result of formalin samples was not interpretable. DNA extracted from formalin-fixed tissue samples is not suitable for oligonucleotide-based a-CGH studies. On the other hand, the molecular fixative preserves tissue DNA similar to its fresh state with no discernable analytical differences.
Introduction
Applications of new technologies have resulted in major advancements in laboratory medicine [1]. Bringing these advances into clinical practice, however, requires careful evaluation and validation. Array-based comparative genomic hybridization (a-CGH) is an extremely powerful tool that can generate high resolution mapping of chromosomal abnormalities. Advances in microarray technology and bioinformatics have now made a-CGH easily available and affordable [14, 15]. With a-CGH’s potential for clinical application, it is important that guidelines for proper sample preparation and control of quality are developed. Since conventional methods of clinical tissue preparation commonly employ formalin fixation, we studied the suitability of formalin for array CGH studies and compared the results to that of clinical samples that were preserved in a newly developed, molecular-friendly fixative.
The study was approved by the University of Miami Institutional Review Board. Three separate normal skin biopsies from one volunteer healthy male were immediately sliced in three parts each 0.1 × 0.2 × 0.2 cm. One part was immersed in normal saline solution, one part in a methanol-based molecular tissue fixative, UMFIX (Universal Molecular Fixative, marketed as Tissue-Tek® Xpress™ Molecular Fixative, Sakura Finetek, Torrance, CA), and the third slice was fixed in 10% neutral buffered formalin. Volume of fixative/preservative was 150 ml and incubation was performed at room temperature. After 8 (set 1), 12 (set 2) and 24 h (set 3), the genomic DNA was extracted from the samples using Puregene DNA Purification System tissue kit (Gentra, Minneapolis, MN). One microliter of extracted DNA solution was diluted in Tris–EDTA (TE) buffer and evaluated on ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE). Gel electrophoresis (0.8% agarose gel) was performed to evaluate the quality of genomic DNA.
DNA yield and quality of genomic DNA as evaluated by spectrophotometry, were similar between the samples (Table 1), although UMFIX samples appeared to have better quality. A260/A280 ratio determines presence of contaminating proteins and a ratio of more than 1.8 is generally considered to be indicative of high quality sample. All UMFIX samples consistently had a ratio of more than 1.8. We further evaluated the quality of DNA by agarose gel electrophoresis that showed presence of high molecular weight (HMW) genomic DNA band in all samples (Fig. 1a). While formalin-fixed sample showed higher degradation and lower intensity of genomic DNA, UMFIX-exposed samples did not appear degraded.
Table 1Spectrophotometric result of total DNA yield, DNA purity, and labeling efficiency, for fresh, formalin-fixed and UMFIX-exposed samplesSampleTotal DNA (μg)A260/A280A260/A230Sample-cy5 labeling efficiency (pmol/μg)Control-cy3 labeling efficiency (pmol/μg)Fresh 13.891.771.4370.8111Fresh 22.941.861.7942.889.6Fresh 32.931.9252.897Mean ± SD3.25 ± 0.551.84 ± 0.071.74 ± 0.2955.47 ± 14.1999.20 ± 10.87UMFIX 16.4161.85278.6110UMFIX 22.71381.861.8449.688UMFIX 34.0121.861.848.281.8Mean ± SD4.38 ± 1.881.86 ± 0.011.88 ± 0.1158.80 ± 17.1693.27 ± 14.82Formalin 15.3661.580.8459.8142.4Formalin 24.04241.671.1141.273.4Formalin 34.4271.932.0836.277.4Mean ± SD4.61 ± 0.681.73 ± 0.181.34 ± 0.6545.73 ± 12.4497.73 ± 38.731 8 h, 2 12 h, 3 24 hFig. 1Result of gel electrophoresis for genomic DNA a HMW genomic DNA is visible in all samples. There is more DNA degradation in formalin-fixed samples. b Formalin-fixed samples do not show uniform strong smear after linear amplification. c PCR for GAPDH shows a 450 bp amplicon in all samples with a lesser intensity in formalin-fixed samples, L ladder (numbers indicate bp), S fresh control in saline, U UMFIX, F formalin, P positive control, N negative control
Genomic DNA from the skin samples and the control female DNA (Promega, Madison, WI) were then subjected to amplification according to Agilent’s (Palo Alto, CA) protocol for oligonucleotide array-based CGH for genomic DNA (version 2.0 August 2005). Amplification, of both male genomic DNA and female control DNA, 100 ng each, was performed using Qiagen REPLI-g Amplification Kit. Amplified DNA was digested during a 2 h incubation at 37°C, with Alu I and Rsa I (10 U/μl; 5μl/reaction) restriction enzymes (Promega, Madison, WI). Purification of digested DNA was performed with QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA). Digested DNA was subjected to electrophoresis to evaluate quality of amplified DNA by visual inspection of its uniformity and range.
The amplification efficiency of DNA from formalin-fixed samples was less than fresh and UMFIX samples, as evidenced by the size and fluorescent intensity of the bands (Fig. 1b).
We also used PCR for GAPDH primers to gauge the quality of extracted DNA. PCR was performed using glyceraldehyde-3-phosphate dehydrogenase primers (GAPDH, Clonetech, Palo Alto, CA) using 0.5 μg of RNase-treated isolated DNA and Qiagen TaqPCR Mastermix (Qiagen, Valencia, CA). The conditions for DNA PCR were as follows: 95°C, 15 min; 35 cycles at 94°C, 45 s; 60°C, 45 s; 72°C, 2 min. As seen in Fig. 1c, a 450 bp band was detected in all samples, although the intensity was considerably lower in formalin-fixed material.
Amplified Genomic DNA was then labeled using BioPrime Array CGH Genomic Labeling kit (Invitrogen, Carlsbad, CA). Quality analysis and quantitation was performed with ND-1000 (NanoDrop Technologies, Wilmington, DE) spectrophotometer to calculate the labeling efficiency. Labeled and purified samples were combined with hybridization master mix and applied to Agilent Human Genome CGH Microarray 44B slides for 40 h at 65°C. To minimize the impact of environmental oxidants on signal intensities, slides were scanned immediately using Agilent microarray scanner 4800B. Array images were analyzed using Agilent feature extraction software (v8.1) and CGH explorer (v2.51) [9]. Microarray analytical variables, including the feature signal intensity, data distribution dynamic range, signal to noise ratio and background intensity levels, and the number of saturated and undetected features was used to assess microarray quality.
One of the steps in microarray quality control is labeling efficacy or dye incorporation. Dye incorporation was not drastically different between fresh and UMFIX-exposed samples but formalin-fixed samples showed less dye incorporation (Table 1). There were no differences between fresh and UMFIX-exposed material with regard to scan parameters (Table 2). Formalin-fixed samples, showed various errors such as over-saturation, non-uniformity in replicates and increased signal to noise ratio. The numbers of non-uniform features were at least ten fold higher in formalin-fixed samples. The signal to noise ratio of replicated probes can be used to evaluate reproducibility of signals. Formalin-fixed samples showed a higher median %CV value, indicating lower reproducibility of signal across the microarray and lower signal to noise ratio. The number of features that were saturated in the scanned image was also significantly higher in formalin-fixed samples.
Table 2Representative data of microarray scan quality measurementSampleNon-uniform featureReproducibility: non-control replicated probes median %CVSaturated featureRedGreenRedGreenRedGreenFresh 11133797801Fresh 2511394400Fresh 3381355600Mean ± SD67 ± 40218 ± 1405 ± 16 ± 200UMFIX 1352560131300UMFIX 2851797800UMFIX 3361366700Mean ± SD158 ± 170292 ± 2338 ± 49 ± 300Formalin 11,4023027762851Formalin 21,98715737123610Formalin 33,1351709483260Mean ± SD2,175 ± 882210 ± 8069 ± 309 ± 3324 ± 380Red cy5 labeled samples, green cy3 labeled controls, 1 8 h, 2 12 h, 3 24 h
These findings may be attributed to erratic and random fragmentation of DNA in formalin-fixed samples. The fragmentation itself results in increase background noise and aberrant signal intensity due to random hybridization.
We further analyzed array scan data by a-CGH explorer software using stem-plot analysis for moving averages. Fresh and UMFIX-exposed sample showed a readable plot with the expected difference in XY chromosome regions; as expected from the female control and male samples. Also, they showed reproducibly amplified and deleted region in our test sample DNA. A-CGH result from formalin-fixed samples was not interpretable (Fig. 2).
Fig. 2Array CGH stem plot of 8-h preserved skin samples—chromosomes are displayed on x-axis and relative ratios on y-axis. Green (control XX female, Cy3 labeled) Red (test XY male, Cy5 labeled). X and Y chromosomes are marked
Array CGH studies have great potential for clinical application. Since the technique does not utilize live cells, it is considerably more advantageous when compared to conventional karyotyping techniques. Array CGH has its own limitations, such as failure to detect translocations; nevertheless it offers unprecedented spatial resolution [6, 11]. Recently, it has been shown that low-level gene copy number change is associated with changes in expression level of its transcripts [12]. Therefore, results of transcriptomics studies can be used to study DNA markers, which are more stable and easier to study than RNA.
Array GGH was originally based on BAC (Bacterial Artificial Chromosomes), but more recently it utilizes oligonucleotides, with its ability to provide whole genome-covering resolution. Synthetic oligonucleotides obviously have the advantage because their exact sequence and length are known for each element on the array. Oligonucleotide array CGH (oa-CGH) has the benefit of overcoming the difficulties inherent to BAC arrays, such as the amount of available DNA, clone management, probe identity due to PCR contaminations, and mapping inaccuracies. Using oa-CGH, it is also possible to eliminate another source of error, which is the batch-to-batch variability of Cot-1 DNA, used to block repetitive DNA sequences, since the oligonucleotide probes are designed to be repeat-free [20].
Samples used in nearly all a-CGH studies have been fresh or fresh-frozen tissue. Such material, although useful in research settings, is impractical and cumbersome to use in clinical practice. Furthermore, because diagnostic biopsy samples are relatively small, the amount of residual tissue for additional ancillary testing may be inadequate. Hence there is a great need to develop test strategies that require minimal amounts of tissue and are robust enough to withstand pre-analytical sample preparation. Simplified schemes for sample preservation that allow reliable histomorphology along with preservation of high quality macromolecules are desirable.
We have previously described a novel tissue fixation and processing technique that beside providing adequate histomorphology also preserves high quality HMW RNA, akin to fresh samples [19]. This fixative also protects HMW tissue DNA for use in PCR studies [18]. Here, we further evaluated the suitability of skin tissue DNA for array CGH studies using same methods applied routinely for fresh samples. This study demonstrates that using the novel fixative it is possible to preserve and extract high molecular weight genomic DNA, supported by high labeling efficiency, comparable to that from fresh tissue. Furthermore, no artifacts were seen using this DNA in microarray scanning or analysis for array CGH.
Array CGH studies have been performed on formalin-fixed tissues with variable and irreproducible results. Most of the prior studies were based on low-resolution BAC arrays without detailed description of DNA quality or array metrics [10, 21]. Besides, there was often inadequate documentation of fixation time, the volumetric ratio of fixative to tissue, or buffering status. Only few studies have adequately addressed analytical aspects of fixation and processing on array CGH. Ghazani et al. [4] studied the effect of formalin fixation on MCF-7 cell line and on one breast cancer tissue sample. They used two different BAC clone arrays; 1.7k for the cell line and 19k for the breast cancer sample [4]. There was no mention of fixative volume or quality of genomic DNA. They showed that long term (1 week) fixation results in extensive loss of HMW DNA. In cell lines, the concordance between fresh and formalin-fixed samples was around 85%. When genomic DNA was amplified, the concordance decreased to 75%.
Johnson et al. [5] also studied the effect of formalin fixation on a-CGH using BAC arrays. Our results support their conclusion that analysis of DNA samples on agarose gels does not offer any advantage in prediction of suitability of formalin-fixed tissue samples for a-CGH. They also show that the quality of a-CGH depends on the integrity of DNA samples with the requirement that extracted DNA supports the PCR amplification of an amplicon of 300 bp or longer. We showed that it is possible to detect amplicons of up to 450 bp in formalin-fixed sample; (albeit with lower intensity when compared to fresh or UMFIX-exposed samples). The studies by Johnson and others show the possibility of having acceptable results with DNA from formalin-fixed samples using BAC arrays but there are no data that supports its suitability for oa-CGH arrays. This may be due to larger sequence of probes presented in BAC array. Other investigators have shown that using alternative approaches to DNA extraction, quantification, amplification or labeling may produce improved results. However, all other authors do agree that the procedures for improving DNA quality are neither substitute for high quality DNA nor they could obtain consistent results from formalin-fixed samples [7, 10]. This is mostly due to complex chemical effect of formalin on tissue, which is still poorly understood. Formalin has a tissue penetration rate of 2.4 mm in 24 h and adequate fixation requires at least a ten to one volumetric ratio of fixative to tissue [3]. Therefore, many clinical specimens are only partially fixed by formalin before processing. Furthermore during processing, they are exposed to formalin and alcohol, introducing formalin and alcohol-related tissue artifacts [13]. Formaldehyde is a dipolar molecule and can react with amino, or imino group of the anionic forms of the amino acids. This reaction is time and temperature dependent [2, 9, 16, 17, 18]. More recent findings show that the deleterious effect of formalin fixation might also result from cumulative action of other reagents and processing conditions. Conversely, no DNA or nucleoside reactions have been reported with ethanol and none would be expected under physiological conditions [8]. By changing the three dimensional structure of the proteins, alcohols prevent protein functions. Therefore, rapid fixation of samples prevents alteration and degradation of biomolecules and preserves them in their native form [11].
In summary, we show that by using a new molecular fixative it is possible to preserve skin tissue DNA that is suitable for array CGH studies; identical to fresh tissue. This can be achieved using same methods and protocols used for fresh samples.

genomics
fixatives
array comparative genomic hybridization
tissue preservation


Intestinal Perforations in Behçet’s Disease

Behçet’s disease accompanied by intestinal involvement is called intestinal Behçet’s disease. The intestinal ulcers of Behçet’s disease are usually multiple and scattered and tend to perforate easily, so that many patients require emergency operation. The aim of this study is to determine the extent of surgical resection necessary to prevent reperforation and to point out the findings of concurrent oral and genital ulcers and multiple intestinal perforations in all patients of our series. During a 25-year study period, information of 125 Behçet’s disease cases was gathered. Among the 82 patients who were diagnosed with intestinal Behçet’s disease, 22 cases had intestinal perforations needing emergency laparotomy. We investigated and analyzed these cases according to the patients’ demographic characteristics, clinical presentations, laboratory data, and surgical outcome. There were 14 men and 8 women ranging from 22 to 65 years of age. Nine cases were diagnosed preoperatively, and the diagnoses were confirmed in all 22 cases during the surgical intervention. Surgical resection was performed in every patient, with right hemicolectomy and ileocecal resection in 11 cases, partial ileum resection in 8 cases with two reperforations, and ileocecal resection in 3 cases with one reperforation.
Introduction
Behçet’s syndrome is a systemic process affecting multiple organ systems1,2. Surgeons need to be aware of the lethal complication of Behçet’s disease with intestinal ulcers, which tend to perforate at multiple sites3,4. A review of the literature reveals that involvement of the gastrointestinal tract is not infrequent. Most cases reported in the literature are in the eastern Mediterranean countries and Japan5–7. We report here a series of 22 cases of intestinal Behçet’s disease with multiple perforations, treated by emergency surgical resections.
Materials and Methods
During the 25 years from July 1979 to June 2004, 125 patients with Behçet’s disease were encountered at the Cardinal Tien Hospital and Tri-Service General Hospital, Taipei, Taiwan. Eighty-two patients were diagnosed as having intestinal Behçet’s disease, which was based on the Mason–Barnes criteria (Table 1)1,2. Among these patients, 22 had intestinal perforations (see Table 2 for the details of these 22 cases).
Table 1The Mason–Barnes CriteriaMajor SymptomsMinor SymptomsBuccal ulcerationsGastrointestinal lesionsGenital ulcerationsThrombophlebitisOcular lesionsCardiovascular lesionsSkin lesionsArthritisNeurologic lesionsFamily historyThree major or two major and two minor criteria are required to establish the diagnosis of Behçet’s diseaseTable 2Intestinal Perforation in Behçet’s Disease Encountered at CTH and TSGH (from 1979 to 2004, n = 22)Case No.Age (years)SexOral UlcerGenital UlcerGI S & SOcular SignsSkin LesionPathergic ReactionArthritis or Arthalgia138M+++−−++245M++++++326F+−++−−447M+++++−528F+−++−++636F++++−−7a22M+++−−+842M++++−922M+++−+++1028F++++−−1165M+++−+−12a23M+++−+−−1332F+−+++−1424M++++−++1534M+++−−1641F+++−+17b38M++++++−1833M+++−+−−1925M+++−+++2048F++++−2129M+++−+−−2250F++++−+Plus signs mean that the feature is present; minus signs mean that the feature is not present.CTH = Cardinal Tien Hospital, TSGH = Tri-Service General Hospital, S & S = symptoms and signsaReperforations at ileum after partial resection of ileumbReperforation at ileum after ileocecal resection
In 13 of these 22 cases, the diagnosis was confirmed at surgical resection for multiple perforations. Nine of the 22 cases had Behçet’s disease with intestinal involvement, which was confirmed preoperatively, six were confirmed by endoscopic examination; two by radiological examination; and one patient had gastrointestinal symptoms of intermittent abdominal pain, diarrhea, and nausea.
Results
Patient Characteristics
There were 14 men and 8 women in the 22 cases investigated. The ages of the patients with perforated intestinal Behçet’s disease ranged from 22 to 65 years, with a mean age of 35.3 years. The age at onset of symptoms of Behçet’s disease varied from 18 to 64 years on diagnosis, with a mean age of 33.1 years.
In Table 2, oral ulcers with gastrointestinal symptoms and signs were found concurrently in all 22 cases, genital ulcers in 19 cases, ocular lesions in 12 cases, and skin lesions in 11 cases. The painful oral ulcers (Fig. 1) occurred on oral mucosa, lips and in the larynx. They varied from 2 to 8 mm in size and invariably healed without scarring. The genital ulcers (Fig. 2) resembled the oral ulcers in appearance and course, except that vaginal ulcers were painless. Four patients had anterior uveitis and eight had a mild relapsing conjunctivitis as their sole ocular lesion. The nodular cutaneous lesions resembled those of erythema nodosum and were chronic and multiple. Most lesions occurred on the chest wall, back (Fig. 3), and legs. Biopsy of dermal subcutaneous lesions had been done in 10 cases. In each of them, a nonspecific vasculitis of subcutaneous capillaries and venules was present (Fig. 4). Pathergic reaction was found positive in 7 of 10 patients.
Figure 1Buccal ulcer.Figure 2Penile ulcer.Figure 3Nodular cutaneous lesion on the back.Figure 4Vasculitis characterized by lymphocytic and plasmacytic infiltration of perivascular tissue (hematoxylin and eosin; 10 × 40).
There were no specific immunologic abnormalities in any of the 16 patients tested (Table 3). The levels of immunoglobulin were variable. IgG was increased in 3 of 16 patients, IgA in 5 patients, and IgM in 3 patients. There was a significant decrease of IgG in two patients and of IgA in one patient. The total hemolytic complement was normal in all 16 serum samples. Alpha-2 globulin was increased in 9 of 16 patients, and gamma globulin was increased in seven patients.
Table 3Laboratory DataCase No.Immunoglobulins (mg/dl)Serum Complement (mg/dl)Globulin (%)IgGIgAIgMC′3C′411,9763752501453813.823.821,726245174924012.010.842,1504002401104514.224.651,500590300382510.518.07a7401856090387.814.381,18019514059326.612.292,2704642621274614.016.2111,8503802501905012.523.512a1,30032023588399.615.0142,3504902951804813.325.01668098561503513.021.817b1,65047528076349.412.5181,8002901501054513.823.2192,4185812091664014.428.0211,8803302501803510.520.0221,9853862281683813.824.2Normal range950–2,110170–41054–26247–19127–524.8–12.18.8–22.8aReperforations at ileum after partial resection of ileumbReperforation at ileum after ileocecal resection
Multiple concurrent penetrating ulcers (Fig. 5) were found in all 22 cases, with multiple perforation sites identified from terminal ileum to the ascending colon (Table 4). The size and number of perforated ulcers were variable, ranging from 0.2 to 6 cm in size, and 4 to 16 in number. The perforations were found at the ileocecal region and ascending colon in 10 cases, at the terminal ileum in 8 cases, and at the cecum and ascending colon in 4 cases.
Figure 5Surgical specimen of ileocecal region showing multiple penetrating ulcers.Table 4Operative Findings and Operation Performed in 22 Perforated Intestinal Behçet’s Disease PatientsCase No.Location of Perforated UlcersNo. of PerforationsOral/genital UlcerOperation Performed1Terminal ileum4+/+Partial resection of the ileum2Terminal ileum6+/+Partial resection of the ileum3Ileocecal region and ascending colon10+/−Right hemicolectomy and ileocecal resection4Ileocecal region and ascending colon16+/+Right hemicolectomy and ileocecal resection5Cecum and ascending colon5+/−Ileocecal resection6Terminal ileum5+/+Partial resection of the ileum7aTerminal ileum4+/+Partial resection of the ileum8Cecum and ascending colon9+/+Right hemicolectomy and ilececal resection9Terminal ileum8+/+Partial resection of the ileum10Ileocecal region and ascending colon11+/+Right hemicolectomy and ileocecal resection11Ileocecal region and ascending colon10+/+Right hemicolectomy and ileocecal resection12aTerminal ileum5+/+Partial resection of the ileum13Terminal ileum7+/−Partial resection of the ileum14Ileocecal region and ascending colon11+/+Right hemicolectomy and ileocecal resection15Ileocecal region and ascending colon5+/+Right hemicolectomy and ileocecal resection16Ileocecal region and ascending colon13+/+Right hemicolectomy and ileocecal resection17bCecum and ascending colon4+/+Ileocecal resection18Ileocecal region and ascending colon7+/+Right hemicolectomy and ileocecal resection19Ileocecal region and ascending colon9+/+Right hemicolectomy and ileocecal resection20Cecum and ascending colon6+/+Ileocecal resection21Terminal ileum4+/+Partial resection of the ileum22Ileocecal region and ascending colon12+/+Right hemicolectomy and ileocecal resectionaReperforations at ileum after partial resection of ileumbReperforation at ileum after ileocecal resection
Operative Treatment and Outcome
All 22 perforated intestinal Behçet’s disease cases were confirmed at operation, with nine of them correctly diagnosed preoperatively. Surgical resection of the perforated intestinal ulcers was done in all cases, with right hemicolectomy and ileocecal resection in 11 cases, partial ileum resection in 8 cases, and ileocecal resection in 3 cases. No reperforation occurred in the group of patients who underwent right hemicolectomy and ileocecal resection. However, two reperforations ocurred in patients who underwent partial ileum resection alone and one in the ileocecal resection group.
The pathologic study of the resected specimens showed nonspecific inflammatory reactions with the infiltration of lymphocytes and plasma cells as the predominant finding (Fig. 6). Histological sections from the ulcer walls showed changes consistent with a nonspecific ulcerative inflammatory process and infiltration containing both plasma cells and chronic inflammatory cells.
Figure 6Chronic inflammatory response and perivascular infiltration (hematoxylin and eosin; 10 × 10).
After operation on these 22 patients with Behçet’s disease and intestinal perforation, four patients died during the postoperative course due to septic shock, which was present prior to the surgical intervention; three died from complications of hypertension and diabetes mellitus; and three were lost to follow-up. Thus, only 12 patients are still under observation, without evidence of gastrointestinal complications up to this date. The remaining 60 cases of intestinal Behçet’s disease, without perforations, are still under surveillance.
Discussion
In 1937, Behçet described a chronic relapsing triple-symptom complex of oral ulceration, genital ulceration, and ocular inflammation5. Over the years, it has become apparent that the process is a systemic recurrent inflammatory disease affecting a number of organs consecutively6. In 1940, Bechgaard first described intestinal involvement in Behçet’s disease. Tsukada et al. proposed the term “intestinal Behçet’s disease” in 19642,3. Baba et al.4. agreed to this proposal and cited 49 cases of the disease treated from 1975. Since then, the number of operations reported has increased rapidly3, but perforated intestinal Behçet’s disease is still rarely reported.
In a large review series, Oshima and colleagues reported that 40% of patients with Behçet’s disease had gastrointestinal complaints, such as nausea, vomiting, and abdominal pain2–4,8,9. The age at onset of these symptoms ranges from 16 to 67 years, and the male-to-female ratio ranges from 1.5:1 to 2:12,5. Our cases were in accordance with this reported age range and sex ratio. The third decade is the most commonly reported age of onset for Behçet’s disease6,8,10,30 and the fourth decade for intestinal Behçet’s disease3. In our study, intestinal Behçet’s disease occurred at a mean age of 33.1 years. However, Behçet’s disease and intestinal involvement were diagnosed simultaneously in some of these patients, most of whom had already experienced systemic manifestations.
The exact cause of this disease still remains an enigma. Current hypotheses include allergic vasculitis of small vessels, autoimmune disease, and immunologic deficiency2,4,11,12. The deposition of immune complexes in the walls of small blood vessels was found by the laboratory results of three of our cases, and this process has been proposed as one of the underlying pathologic mechanisms in intestinal Behçet’s disease12.
Since no clinicopathologic findings are pathognomonic in this disease, the diagnosis is made on the basis of combinations of various clinical symptoms and signs13. Mason and Barnes constructed an elaborate set of major and minor criteria for diagnosis1. They suggested the triad of buccal ulceration, genital ulceration, and eye lesion and skin lesion as major symptoms. The minor symptoms included gastrointestinal lesions, arthritis, thrombophlebitis, cardiovascular lesions, neurologic lesions, and family history. Three major criteria or two major criteria and two minor criteria are necessary for diagnosis. These various symptoms are not usually present at the same time. If we hold the original triple-symptom complex as a prerequisite for the diagnosis, cases may be missed. In 1990, the International Study Group for Behçet’s Disease14 introduced a diagnostic criteria requiring the presence of oral ulcerations plus any two of the following: genital ulcerations, typical eye lesions, typical skin lesions, or positive results to a pathergy test. However, some reports have shown that almost 20% of patients with Behçet’s disease presented without oral lesions initially15,16. Furthermore, 2–5% of patients did not show any oral lesions at all16,17. In our series, all patients had manifestations of concurrent oral ulceration. All perforated cases present oral or genital ulcerations at the same time. Because we warned that patients of intestinal Behçet’s disease may have abdominal pain and oral or genital ulcerations concurrently, intestinal perforations should always be kept in mind.
A phenomenon of pathergy was first described by Blobner in 1937 and was further elaborated by Katzenellenbogen in 1968. It consists of an itradermal test applied to Behçet’s disease patients with a sharp needle prick causing skin hypersensitivity, which is characterized by the formation of a sterile pustule 24 to 48 h after the trauma. Biopsy at the intradermal puncture site is taken 48 h after for histopathologic evaluation. In a study conducted by Tuzum et al., this reaction was found to be positive in 84% of 58 patients with the disease, as compared to 3% of 90 healthy controls1. A positive pathergic reaction should make us aware of the possibility of the disease in the presence of any of the accepted symptoms of this process. However, the recent results and interpretations of pathergy tests have varied widely according to the technical aspects of the tests18,19 and ethnic differences of the patients.
The histological lesions in Behçet’s disease are rather uncharacteristic. Nonspecific perivascular infiltrations of plasma cells and lymphocytes are usually found in the cutaneous and mucosal lesions5,20. The intestinal ulcers in Behçet’s disease are characterized not only by the absence of the granulomatous formation of Crohn’s disease, but also by deeper penetration of the ulcers to areas nearer to serosa membrane than the ulcers of ulcerative colitis3,4,21. The ulcers tend to be undermined, and the submucosal connective tissues are usually destroyed. The bases of the ulcers are avascular with edema-like swelling and crater-shaped formation around the ulcer margin.2,22–24 These ulcers are usually found in the terminal ileum and the cecum, but they may be present at any site throughout the digestive system and tend to perforate at multiple sites25–29. The gross pathologic characteristics of our intestinal Behçet’s disease included perforations at multiple sites concurrently in variable sizes and configurations, extending from the ileocecal region to ascending colon, in accordance with the reported literature3,4,8,30,31.
The medical treatment of the intestinal Behçet’s disease remains unsettled. The beneficial effect of steroid therapy has not been convincing in most series2,7,30. It may control the disease initially, but recurrences are common. Topical application of corticosteroids decreases the ocular inflammation, and is also useful in relieving the pain of oral ulcers. Haim and Sherf reported a favorable response to fresh blood and plasma in cases of Behçet’s disease, but the nature of the useful component in hematotherapy is unknown5. In our two patients with perforations, steroid therapy was given for 2 weeks after surgery with favorable outcomes.
Resection of the ileocecal region or the right half of the colon is the usual operation in the treatment of gastrointestinal complications3,4. In our series, perforations at multiple sites were found in all cases; right hemicolectomy and ileocecal resection were performed in 11 cases without reperforation; ileocecal resection in 3 cases with one reperforation; and partial resection of the ileum in 8 cases with two reperforations.
Conclusion
Because concurrent oral and genital ulcers were found in all patients in our series, the presentation of this seemingly innocuous clinical manifestation along with gastroinstestinal symptoms should raise the level of suspicion that intestinal involvement and complications of perforations may have already happened. The other constant finding among our 22 patients is that all the intestinal perforations were located between the terminal ileum and the ascending colon. Therefore, to prevent reperforations, wide excision of the terminal ileum with right hemicolectomy is recommended for perforated intestinal Behçet’s disease. We found out that the specimens of the resected bowel of the 19 nonreperforated patients all had more than 60 cm of terminal ileum, but those of the three reperforated cases had less than 60 cm. Furthermore, the perforation sites were all at 10 to 12 cm proximal to the anastomosis. This is the main reason we recommend the resection of up to 80 cm of ileum from the ileocecal valve at the time of right hemicolectomy4,31.

intestinal perforations
behçet’s disease
intestinal ulcers


Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow☆

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14 ± 0.4 postpartum (n = 12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling.
1
Introduction
In dairy cows, the peri-partum period is critical to future milk production and fertility. Uterine involution involves extensive restructuring of the extracellular matrix alongside mitogenesis and apoptosis [1–3]. Initial degeneration of placental cotyledons and maternal caruncles accumulate as tissue debris in the uterine lumen forming a lochial discharge [4]. Contractions of the myometrium aid expulsion of lochia, and also restore uterine size, shape and tone to that of a non-pregnant animal [5,6]. Whilst most of these changes have occurred within 2–3 weeks postpartum, involution is not considered complete until about 40–50 days postpartum [1]. The previously non-gravid uterine horn returns to a non-pregnant state 10–15 days earlier than the previously gravid uterine horn [7]. Histological repair of the endometrium lags physical involution by 10–20 days [8], completing when caruncles regenerate epithelium [4]. Microbial contamination of the postpartum uterus is almost universal during the first week postpartum [9]. When pathogenic bacteria are not cleared the uterus becomes infected and inflamed and uterine involution is delayed [1,10]. Clinical endometritis is characterised by the continued presence of a purulent discharge beyond 21 days after calving [1].
Many processes involved in uterine repair are common to those of wound healing in other tissues (for a review see [11]). Potential mediators of tissue turnover and remodelling in the uterus include cytokines, matrix-degrading enzymes and growth factors [11,12]. The insulin-like growth factors (IGF-I and IGF-II) function in such tissue repair processes. In healing-impaired wounds, the mRNA for IGF-I, IGF-1R, and IGFBP-3 is significantly reduced [13]. The administration of IGF-I to these wounds corrects defective tissue repair [14] and in combination with other growth factors it increases connective tissue regeneration and epithelialisation [15]. Components of the IGF system have been described in the uteri of a variety of species (e.g. humans [16], rodents [17], pigs [18], cattle [19], and sheep [20]). The proliferative and differentiating effects of IGFs on uterine cells are thought to support the growth and regression of uterine tissue throughout the estrous cycle and also the regenerative processes in women following menstruation [16,21]. IGFBP-2 has also been shown to stimulate endometrial cell mitogenesis directly [22].
An increased rate of uterine involution is associated with earlier resumption of ovarian activity [23], which is in turn important for increasing pregnancy rate to first service [24]. Conversely, endometrial damage associated with sub-clinical endometritis leads to prolonged intervals to conception, with many cows failing to conceive at all [25]. The mechanisms that regulate uterine involution are not completely understood and, to the best of our knowledge, no previous studies have investigated the uterine IGF system during involution in lactating dairy cows. We postulated that changes in IGF bioavailability may be implicated in the rate of postpartum uterine recovery and thus influence the calving to conception interval and reproductive efficiency. The objective of the study was to determine patterns of mRNA expression for the IGF system within the previously gravid (PG) and previously non-gravid (PNG) uterine horns during the early postpartum period. Samples were obtained at approximately 2 weeks after calving as we hypothesised that this represents a time by which a delay in the normal recovery process may predispose cows to the subsequent development of endometritis.
2
Materials and methods
2.1
Animals and tissue samples
All procedures were carried out under license in accordance with the European Community Directive, 86-609-EC. Uteri were collected from 12 multiparous Holstein-Friesian dairy cows (mean parity 4.7) following slaughter at day 14 ± 0.4 postpartum. The diameters of both horns were measured approximately 5 cm anterior to the bifurcation of the uterus. Samples of inter-caruncular and caruncular tissue were dissected from the previously gravid and non-gravid uterine horns approximately 1 cm anterior to the bifurcation of the uterus. A 5 cm square region was harvested, wrapped in aluminium foil, and frozen in liquid nitrogen-tempered isopentane. Samples were stored at −80 °C until sectioning.
2.2
In situ hybridisation
The in situ hybridisation procedure was performed as described previously [26]. All chemicals were purchased from Sigma–Aldrich Company Ltd. (Poole, Dorset, UK) or VWR International Ltd. (Poole, Dorset, UK) unless otherwise specified. Briefly, sections of 10 μm were cut from each uterine tissue sample and thaw-mounted onto SuperFrost® Plus or POLYSINE™ microscope slides, fixed in 4% (w/v) paraformaldehyde in 0.01 M PBS, washed in PBS, and sequentially dehydrated in 70% and 95% ethanol. The oligonucleotide probes for the IGF system were end-labelled with [35S]dATP (Amersham Biosciences UK Ltd., Buckinghamshire, England) using terminal deoxynucleotidyl transferase (Promega UK Ltd., Southampton, England). Tissue sections were subsequently treated with 100 000 cpm (100 μl)−1 hybridisation buffer and hybridised overnight at either 42, 45, or 52 °C (Table 1). Following incubation, slides were washed in a solution of 1 × SSC, 2 g l−1 sodium thiosulphate at room temperature for 30 min followed by fresh 1 × SSC, 2 g l−1 sodium thiosulphate at 60 °C for 60 min. Slides were then rinsed in solutions of 1 × SSC, 0.1 × SSC, 75% ethanol and 95% ethanol and air-dried before exposure to β-max hyperfilm (Kodak BioMax MR Film) for either 4 or 5 days. All uterine sections treated with a particular probe were hybridized in the same batch. Sense probes, which were identical in sequence to the respective mRNA targets, were always included as negative controls and any signal from these was regarded as non-specific. Each batch also contained an appropriate positive control tissue, based on previous studies. These were cross-sections of uterus from an estrous ewe for IGF-I and the type 1 IGF receptor [20], IGFBP-1 [27] and IGFBP-6 [28]; ovine placentome for IGF-II and IGFBPs-2, -3 and -4 [29] and ovine intercotyledonary tissue for IGFBP-5 [30].
2.3
Photographic emulsions
To aid cellular localisation of hybridised probes, slides previously subject to autoradiography were coated with photographic emulsion LM1 (Amersham Biosciences UK Ltd., Buckinghamshire, England) according to the manufacturer's instructions and stored for 28, 30 or 42 days at 4 °C in the dark (Table 1). The slides were developed in 20% phenisol (ILFORD Imaging UK Ltd., Cheshire, England) fixed in 1.9 M sodium thiosulphate and counterstained with haematoxylin and eosin. All other slides were also stained with haematoxylin and eosin to aid identification of tissue region.
2.4
Optical density measurements
Readings were obtained from at least two sections per tissue for each of the antisense (AS) and sense (S) probes. Autoradiographs were scanned into a computer and optical density (OD) measurements were recorded from digital images. The relative expression of mRNA for components of the uterine IGF system was quantified from the autoradiographs using the public domain NIH ImageJ program (available through the NIH website—http://www.nih.gov), which calculated the average optical density (OD) over the selected area of film based on a linear grey scale of 0.01–2.71. The following tissue layers were each assessed separately: luminal epithelium, sub-epithelial stroma (a layer of dense connective tissue underlying the luminal epithelium), caruncular stroma (the dense connective tissue forming the caruncles), deep endometrial stroma (loose connective tissue between the sub-epithelial stroma and the myometrium) and myometrium. The latter two tissue types were only present in samples collected from the inter-caruncular region. Each tissue type was measured separately on each section. The background OD, from a blank area of film, was also measured and subtracted from both AS and S OD measurements. Finally the S values were subtracted from AS values to give an average OD value for specific hybridisation [31]. The detection limit was taken as an OD value of 0.01.
2.5
Statistical analysis
Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS for Windows, V13.0). Data for uterine diameter measurements at the time of tissue collection were analysed using Student's t-test. OD measurements were obtained from four samples per cow, taken from each of the caruncular and inter-caruncular regions of the previously gravid and non-gravid horns. The effects of uterine horn and tissue region on the level of mRNA expression for each probe were analysed by general linear model analysis. Cow was entered as a random effect. For this purpose, data from uteri in which a particular probe showed no detectable specific hybridisation (OD of <0.01) were given an OD of 0.01, which equated to the lower limit of detection. Results were considered statistically significant when P < 0.05.
3
Results
At the time of tissue collection, the diameter of the previously gravid uterine horn was larger than that of the previously non-gravid uterine horn (56 ± 6.9 and 31 ± 3.1 mm, respectively, mean ± S.E.M., P = 0.005). The spatial distribution of mRNA encoding components of the uterine IGF system is shown in Figs. 1 and 2. The concentrations of mRNA in OD units are summarised in Table 2 according to uterine horn and tissue region and their two-way interactions are illustrated in Figs. 3 and 4. The method used provided a semi quantitative measure of the intensity of mRNA expression in specific cell types.
3.1
Expression of the IGFs and IGF type 1 receptor
IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium in both uterine horns (Figs. 1(A) and 2A). Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma (not shown) and myometrium (Figs. 1(C), (E) and 2C, E).
Overall expression of IGF-I mRNA was higher in the inter-caruncular than caruncular SES (P = 0.001, Table 2). There was a significant horn × region interaction (P = 0.032), with lower levels of IGF-I transcript in the inter-caruncular SES of the PG compared with the PNG horn (Fig. 3(A)). IGF-II expression was higher in the DES than in the caruncular stroma and myometrium (P ≤ 0.001, Table 2). When data from tissue regions were pooled the concentration of IGF-II mRNA did not vary between the PNG and PG horns, but there was a significant horn × region interaction (P ≤ 0.001). Expression of IGF-II in the DES and caruncular stroma was lower in the PG than PNG horn, whereas within myometrium expression was higher in the PG than PNG horn (Fig. 3(B)). For the IGF-1R, expression was highest in myometrium and similar between DES and caruncular stroma (Table 2 and Fig. 3(C)). Overall, the level of IGF-1R transcript was higher (P = 0.030) in the PNG than PG horn (Table 2). The horn × region interaction was not significant for uterine IGF-1R mRNA expression.
3.2
Expression of IGFBPs
IGFBP-1 mRNA could not be detected in any uteri examined, despite expression being observed in the ovine estrous uterus which was used as positive control tissue (data not shown). IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma (Figs. 1(G), (K), (O) and 2G, K, O). IGFBP-4 mRNA was additionally expressed in myometrium. In contrast, IGFBP-3 mRNA expression was only detected in the luminal epithelium (LE) of both inter-caruncular and caruncular samples (Figs. 1(I) and 2I). IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma (Figs. 1(M) and 2M).
IGFBP-2 mRNA expression in inter-caruncular and caruncular SES was higher than in DES and caruncular stroma (P ≤ 0.001, Table 2). There was no main effect of horn, but there was a horn × region interaction (P = 0.034). Within caruncular stroma only, the concentration of IGFBP-2 mRNA was higher in the PG than the PNG uterine horn (Fig. 4(A)).
For IGFBP-3 mRNA the main effects of uterine horn and tissue region were not significant but there was an interaction (P ≤ 0.001). Expression in the inter-caruncular LE was higher in the PNG than PG horn, whereas in the caruncular LE expression was higher in the PG uterine horn (Fig. 4(B)).
Expression levels of IGFBP-4 mRNA varied between tissue regions, with higher expression in the caruncular than inter-caruncular SES, lowest expression in myometrium, and intermediate signal intensity in the DES and caruncular stroma (Table 2). There was no difference in transcript levels between the PNG and PG uterine horns when regional data were combined (Table 2). Levels of IGFBP-4 mRNA expression were, however, affected by an interaction between uterine horn and tissue region (P = 0.024): within DES expression was lower in the PG than PNG uterine horn (Fig. 4(C)).
Expression of IGFBP-5 mRNA was highest in myometrium, intermediate in the inter-caruncular and caruncular SES and lowest in caruncular stroma (P ≤ 0.001, Table 2). When regional expression data were pooled, the PNG uterine horn expressed higher concentrations of IGFBP-5 mRNA than the PG horn (P ≤ 0.001, Table 2). There was also a significant effect of the interaction between uterine horn and tissue region (P ≤ 0.001). Expression in both the inter-caruncular SES and the caruncular stroma was lower in the PG than PNG uterine horn whereas for the caruncular SES the reverse was true (Fig. 4(D)).
IGFBP-6 mRNA was expressed at higher concentrations in the inter-caruncular and caruncular SES than in DES and caruncular stroma (P ≤ 0.001, Table 2). Transcript levels were higher (P ≤ 0.001) in the PNG than PG uterine horn when regional expression data were pooled (Table 2). The interaction between uterine horn and tissue region was significant (P = 0.045). Expression in each of the inter-caruncular SES, caruncular SES, DES and caruncular stroma was lower in the PG than PNG horn (Fig. 4(E)).
4
Discussion
The rate of uterine involution is an important factor influencing the subsequent fertility of dairy cows [24]. In this study we have investigated for the first time a possible role for the IGF family of proteins in this event in lactating dairy cows. The timing of tissue collection at approximately day 14 postpartum occurred when the PG uterine horn in our group of multiparous cows was larger than the PNG horn. At this stage caruncular tissue is expected to have undergone degeneration and sloughing, but not to have completed re-epithelialisation [2]. In contrast, the inter-caruncular area does not lose its epithelial layer [32] and recovers from pregnancy more quickly [2]. The ongoing process of uterine involution at the time of tissue collection was thus expected to involve tissue regeneration alongside size recovery. An adequate recovery process may be crucial in preventing the uterus, which is heavily contaminated with bacteria following calving [1], from developing endometritis. Samples were analysed using in situ hybridisation. Whilst this approach is considered only semi-quantitative, we have found the technique described here to be highly repeatable. Furthermore, it enables measurement of mRNA concentrations in individual cellular types. This is not feasible in a complex organ such as the uterus using alternative techniques such as RT-PCR, as it is not readily possible to separate different populations of epithelial and stromal cells for RNA extraction.
IGF-I mRNA was localised to the SES, confirming earlier observations in the cow [33] and sheep [20,34]. Normal wound healing involves a sequence of inflammation, proliferation, and maturation or remodelling [12] and local IGF-I production increases as wound healing progresses [35]. Since IGF-I increases during the late proliferative phase of the human menstrual cycle [36], and is known to stimulate cell proliferation and collagen synthesis during tissue regeneration [14,35], we propose that IGF-I produced by SES may act in an autocrine and/or paracrine manner to stimulate the proliferation of uterine stroma and epithelium [37,38] during uterine involution.
In early pregnancy the bovine endometrium synthesises IGF-II primarily within caruncular stroma [33]. The present study localised IGF-II mRNA at similar concentrations in both the caruncular stroma and myometrium. The strongest expression of IGF-II mRNA was, however, in the DES. Similar results were found in human endometrium [36]. IGF-1R was similarly localised to the DES, caruncular stroma and myometrium, confirming earlier observations in the bovine uterus [33]. Since the effects of IGF-II are probably mediated by the IGF-1R (for a review see [39]), the co-localisation of IGF-II and IGF-1R transcripts supports a local action for IGF-II in uterine repair and regeneration within both endometrial and caruncular stroma [16,21]. Stromal IGF-II may also act in a paracrine manner to stimulate epithelial cell proliferation [22]. The interaction between IGFs and their receptors in muscle growth and regeneration has been comprehensively reviewed by Florini et al. [40]. In myometrium, IGF-II may stimulate muscle growth and regeneration [40], and potentially increases muscle strength [41]. These actions would support myometrial contractions that return the uterus to its non-pregnant size, shape and tone [5]. Since the PG horn has to contract from a larger size at parturition, the higher concentration of IGF-II in the PG myometrium supports the proposal that IGF-II assists postpartum uterine size recovery. In rat myometrium the IGF-1R is up-regulated in the early postpartum period [42].
This study failed to detect IGFBP-1 mRNA in any postpartum uteri. During the estrous cycle IGFBP-1 expression is low at estrous and relatively higher during the luteal phase [33] concurrent with progesterone production. Since the cows in this study had yet to establish ovulatory cycles, then the uterus would not have been recently exposed to progesterone stimulation [43]. In ruminants IGFBP-1 appears to be involved in pregnancy recognition [33,44] rather than, as this study shows, postpartum uterine events.
In agreement with previous studies [33,44], the expression of uterine IGFBP-2 mRNA was localised to the SES and at relatively lower levels in the DES and caruncular stroma. In the cyclic cow IGFBP-2 mRNA levels increased during the luteal phase, concomitant with the highest levels of plasma progesterone [19]. Other studies have shown that human endometrial cells constitutively synthesise and secrete IGFBP-2 in vitro
[45] and in response to estradiol [46]. In the bovine mammary gland [47], IGF-I may stimulate IGFBP-2 expression and protein secretion, whereas in fetal visceral glomerular epithelial cells isolated from human kidneys IGFBP-2 production was stimulated by IGF-II [48]. The exact mechanisms regulating postpartum uterine IGFBP-2 mRNA expression thus requires further investigation. IGFBP-2 is presumably modulating uterine involution indirectly by regulating the bioavailability of IGF-I and IGF-II and the interaction of these ligands with their receptors [39]. The precise action of IGFBP-2 also remains uncertain. Both stimulatory [22] and inhibitory [48] actions of IGFBP-2 on IGF-stimulated epithelial cell proliferation have been suggested. Alternatively or additionally, IGFBP-2 could modulate uterine cell growth directly [22].
In contrast to all the other binding proteins investigated, the expression of IGFBP-3 mRNA was confined to the luminal epithelium, again agreeing with earlier work in the cyclic animal [33]. Epithelial IGFBP-3 may regulate local IGF bioavailability [39] or transport IGFs across this cell layer [49,50] for secretion into the uterine lumen. Removing excess IGF from the endometrium would prevent the IGF-1R from being down-regulated [51]. Alternatively, since IGFBP-3 associates with cell surfaces, then it may store IGFs [52] in the uterus and further promote IGF-stimulated tissue repair [37,39] as proposed for other physiological systems [53].
IGFBP-4 mRNA was detected in multiple uterine tissue compartments. The localisation in SES and caruncular stroma has also been found in the pregnant ewe [54] and synthesis in the DES and myometrium agrees with studies in the human and pregnant bovine uterus [44,55]. IGFBP-4 is generally considered inhibitory to IGF actions [56]. IGFBP-4 does not appear to bind to the cell surface or extracellular matrix, but can cross the endothelium [39], indicating that IGFBP-4 may clear endometrial IGFs.
IGFBP-5 mRNA was localised to caruncular stroma and myometrium, in agreement with previous studies in the cow [33] and sheep [34], and transcript was also detected in the SES. With a lack of detectable IGFBP-2, IGFBP-3, and IGFBP-6 mRNA alongside relatively low levels of IGFBP-4 mRNA in the myometrium, the abundance of IGFBP-5 in this tissue suggests this binding protein as the primary regulator of local IGF bioavailability [57]. In the rat myometrium, IGFBP-5 mRNA is significantly up-regulated after parturition, which is suggested to support tissue remodelling during involution [42]. It is also possible that within the myometrium, IGFBP-5 is directly stimulating muscle cell survival during myogenesis [57,58]. In stromal fibroblasts, IGFBP-5 can adhere to the extracellular matrix, which decreases its affinity for IGF and can potentiate IGF-stimulated DNA synthesis [39,59]. Furthermore, IGFBP-5 may stimulate local tissue growth independently of IGF [39,59].
IGFBP-6 was localised to SES and at lower levels in DES and caruncular stroma, similar to the non-pregnant ovine uterus [28]. This expression pattern parallels that of IGFBP-2 mRNA and may suggest these two binding proteins are co-regulated [60]. IGFBP-6 has a markedly higher affinity for IGF-II [61] and so the major function of IGFBP-6 is probably to regulate IGF-II actions [62,63]. Furthermore, IGFBP-6 is generally considered to inhibit the effects of IGF-II, including cell proliferation and differentiation [59]. The importance of controlling uterine IGF bioactivity has been demonstrated in the human uterus where low levels of IGFBP-6 and higher levels of IGF-II are associated with uterine leiomyomas (fibroids), compared with normal endometrium [64].
Many members of the IGF family showed differential expression between the two uterine horns. Expression of IGF-I, the IGF-1R and IGFBP-6 was at an overall lower level in the previously gravid horn whereas IGFBP-4 mRNA expression was lower in the DES only. IGF-II, IGFBP-3 and IGFBP-5 expression showed horn by region interactions, with mRNA concentrations reduced for some regions but increased for others. These differences may reflect the temporal misalignment of the horns in their rate of tissue remodelling, with the PG horn lagging behind the PNG horn by up to 15 days [7].
In conclusion, the IGF system is significant to uterine function and the synthesis of these growth factors in the postpartum uterus indicates a role in uterine involution. This study has shown that in the postpartum bovine uterus IGF-I synthesis was localised to sub-epithelial stroma, whilst maximum concentrations of IGF-II and IGF-1R mRNA were in the endometrial stroma and myometrium, respectively. The uterine tissue compartments expressed different profiles of IGF-binding proteins, indicating that IGF bioavailability and bioactivity is differentially regulated throughout the regenerating endometrium. IGFs are presumably supporting the tissue repair that follows parturition, similar to that of normal wound healing [12]. We propose that myometrial IGF-II synthesis stimulates tissue recovery in an autocrine manner, which may assist the uterus returning to its non-pregnant shape and size. Although IGFs may be key physiological mediators of endometrial repair, other growth factors and cytokines are undoubtedly also important in this process [11,65].
Conflicts of interest
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


The Use of Herbal Medicine in Alzheimer's Disease—A Systematic Review

The treatments of choice in Alzheimer's disease (AD) are cholinesterase inhibitors and NMDA-receptor antagonists, although doubts remain about the therapeutic effectiveness of these drugs. Herbal medicine products have been used in the treatment of Behavioral and Psychological Symptoms of Dementia (BPSD) but with various responses. The objective of this article was to review evidences from controlled studies in order to determine whether herbs can be useful in the treatment of cognitive disorders in the elderly. Randomized controlled studies assessing AD in individuals older than 65 years were identified through searches of MEDLINE, LILACS, Cochrane Library, dissertation Abstract (USA), ADEAR (Alzheimer's Disease Clinical Trials Database), National Research Register, Current Controlled trials, Centerwatch Trials Database and PsychINFO Journal Articles. The search combined the terms Alzheimer disease, dementia, cognition disorders, Herbal, Phytotherapy. The crossover results were evaluated by the Jadad's measurement scale. The systematic review identified two herbs and herbal formulations with therapeutic effects for the treatment of AD: Melissa officinalis, Salvia officinalis and Yi-Gan San and BDW (Ba Wei Di Huang Wan). Ginkgo biloba was identified in a meta-analysis study. All five herbs are useful for cognitive impairment of AD. M. officinalis and Yi-Gan San are also useful in agitation, for they have sedative effects. These herbs and formulations have demonstrated good therapeutic effectiveness but these results need to be compared with those of traditional drugs. Further large multicenter studies should be conducted in order to test the cost-effectiveness of these herbs for AD and the impact in the control of cognitive deterioration.
Introduction
Alzheimer's disease (AD) is characterized as a progressive neurodegenerative disorder and considered as prominent cause of dementia in the elderly. The main characteristics of this disease are difficulties in household handling routine and cognitive and emotional disturbance in the elderly. The treatment of AD is a clinical challenge. With the development of cholinesterase inhibitors and a N-methyl-d-aspartate antagonist (memantine), good perspectives have emerged in controlling the symptoms of AD. Therapeutic decisions have to be guided by clinical studies and should consider the physiopathogenesis and epidemiology of the disease.
The main objective of these clinical trials is to reduce the Behavioral and Psychological Symptoms of Dementia (BPSD) and to improve cognition and the functional activity status, thus reducing the impairment of instrumental activities of the daily living (IADLs) and to lower the institutionalization rates (nursing home placement). Unfortunately, only a limited number of trials have dealt with this topic and with follow-up periods shorter than two years. In spite of the absence of sufficient therapeutic effectiveness in mild and moderate AD, these drugs are still considered as the first line of treatment for AD (1). Studies of cost-effectiveness suggest that memantine (2,3) and donepezil (4) are useful in the reduction of institutionalized care and/or cognitive impairment in patients with AD. Recently, two clinical trials showed no improvement of the cognitive deficit (5,6) or reduction in the institutionalization rate (6).
Searching for alternatives, many herbal products have been tested and employed in the treatment of AD, but with different clinical responses (7). The assessment of these drugs through randomized controlled trials should be useful to identify effective products in the treatment of AD.
Methods
Searching at MEDLINE (during April 2006, PubMed), LILACS (Latin American and Caribbean Health Science Literature: 40th edition, May 2001, the last research was performed in April 2006); Cochrane Library (issue 1, 2006); Dissertation Abstract (USA, during April 2006); ADEAR (Alzheimer's Disease Clinical Trials Database, until April 2006); National Research Register (1/2006); Current Controlled trials (the last research was performed in October 2005); PsychINFO Journal Articles (during the year of 2006); relevant web sites; and scanning of reference list of relevant articles. There were no language or publication restrictions.
Search for keywords in MeSH (medical subject heading (MeSH) with the words ‘Alzheimer disease, dementia, cognition disorders’ was performed first. In the second part, the keywords were ‘Herbal’ and ‘Phytotherapy’. The crossover results of the two searches were evaluated by the Jadad's measurement scale (8).
Inclusion criteria: Three investigators independently reviewed all of the articles found. The articles were selected using the criteria listed below:
The studies should be randomized; double-blind and controlled (with a control group and a treatment group).
Studies should establish methodological procedures in the crossover or be conducted at the same time.
In the case of being crossover, a washout period of at least 7 days was required.
Patients included in the researches had their diagnosis rated into three degrees as follows: mild, moderate and severe forms of AD, according to the criteria from the National Institute of Neurological and Communicative Disorders and Stroke—AD and Related Disorders Association (NINCDS-ADRDA) (9). The models used were as follows: Mini-mental beginning values between 10 and 26 (initial and mild group) or <10 (initial group).
Clinical trials should last for at least 1 month (4 weeks).
Detailed description of the herbal product used.
Neuropsychiatry symptoms progression should be measured with numerical score using the Assessment Scale (ADAS-noncog, range of score, 0–70), NPI (Neuropsychiatric Inventory, range of score 0–120), Clinical Global impression of change or Behavioral rating scale for Geriatric patients.
The final score should be quantified using a combination of ADL and IADL methodological procedures.
Exclusion criteria: The herbal product has already been target of a quantified systematic review study. In this case, only the results of the studies will be considered.
Jadad's measurement scale: Methodological quality was assessed using a scale developed and validated by Jadad et al. (8). This scale assesses the completeness of reporting using three items with a five points maximum score. If the allocation into groups is explicitly randomized, item 1 is scored. A bonus point is given if an adequate method to generate the random sequence is described. If there is an explicit statement that the study is double-blind Item 2 is scored. A bonus point is given if the method is described and adequate. Item 3 is scored if there is either an explicit statement that all patients included were also analyzed or if the number and reasons for dropouts in all groups are given separately. For being classified as adequately reported a trial should score at least three of five points, a cut-off point is recommended by the author of the scale (10).
All extraction and quality assessments were performed by at least two independent reviewers using standard forms developed for each review. Disagreements were documented and discussed with final decisions made by the principal reviewer.
Results
Two herbs and two herbal formulations were identified to have effectiveness in the treatment of cognitive disturbance of AD in the systematic review: Salvia officinalis (11), Melissa officinalis (12), and Yi-Gan San (13) and Ba Wei Di Huang Wan (BDW) (14). The main characteristics of the study are described in Table 1.
Gingko biloba was previously identified in one meta-analysis (15), and only the conclusions of the study will be considered. Another study will be conducted with huperzine A, a product derived from a Chinese herb Huperzia serrata, to evaluate the safety and efficacy in the treatment of AD in a multicenter randomized controlled trial of its effect on cognitive function (16).
The studies of Salvia (11), Melissa (12), Yi-Gan San (13) and BDW (14) have reached Jadad's measurement scale of ≥ 3. The researches had a follow up of 1 month (Yi-Gan San) (13), 2 months (BDW) (14) and 4 months (Salvia and Melissa) (11,12). All samples studied were composed of patients with initial mild symptoms judged as AD. Two studies compared herbal medicines and control samples, using intention to treat [Salvia (11) and Melissa (12)].
None of the studies evaluated the institutionalization rate or compared the active principle with the current therapies with Acetyl Cholinesterase Inhibitor or memantine.
Discussion
The results of this systematic review identified four studies with methodological quality assessing S. officinalis (11), M. officinalis (12), Yi-Gan San (13) and BDW (14). The last two are composed of formulations with different phytoactive agents. These herbs and formulations presented efficiency in reducing the mild and moderate symptoms of AD.
Gingko biloba presented statistically significant mild effectiveness in the treatment of cognitive deficit in AD. The meta-analysis study of Cochrane (13) concluded that additional controlled studies would be necessary in order to recognize cognitive improvement with the use of gingko. There is still need of a prospective study with an appropriate duration and representative sample to identify if G. biloba reduces the development of AD (17). Another plant with a large application perspective is H. serrata, after multicenter trial confirmation underway (16).
Melissa and Yi-Gan San showed reduction in the cognitive deficits and a good sedative effect in patients with AD (12,13). Previous clinical studies showed that the extract of M. officinalis reduces laboratory-induced stress (18) and might have benefits in mood improvement (19–21). The use of these herbs and formulations should be well tolerated, (22) and adverse effects have not yet been reported (23). Further studies should be conducted to compare the current therapies for AD and the use of these herbal remedies in controlling the symptoms of AD.
The action mechanisms of these herbs and formulations are not well known. It has been suggested that the chemical composition of the essential oil of the Melissa and Salvia leaf extracts are monoterpene aldehydes, polyphenol flavonoids (including rosmarinic acid) (24) and monoterpene glycosides (25). All of these components have many observable effects in vitro, which include powerful anti-oxidative activity (26,27) and an affinity to nicotinic and muscarinic receptor in the human cerebral cortex (28). This last mechanism is of special interest, as modulation of cholinergic systems should play a role in improving the cognitive function, especially in AD.
Yi-Gan San and BDW are mixes of many herbal ingredients. Yi-Gan San is a mixture of 7 different dried plants, many of them (Unticariae sinensis and Angelicae root) with possible actions in the serotoninergic and gaba system. BDW consists of 8 herbs: Rehmannia glutinosa Libosh. var purpurea Makino, Cornus officinalis Sieb et Zucc (Cornaceae), Dioscorea batatas Decne root (Dioscoreaceae), Alisma orientale Juzep rhizome (Alimataccae), Poria cocos Wolf, Paeonia suffruticosa Andr. (Paeoniaceae), Cinnamomum cassia Blume (Lauraceae) and Aconitum carmichaeli Debx. (Ranunculaceae). Studies have suggested that BDW enhances the choline acetyltransferase activity and increases the acetylcholine content of the frontal cortex in a murine model (29,30).
Major methodological limitations of the four studies are small size of samples and short-term duration. There is no description of the chemical composition and/or possible active principles of the different products employed in studies Involving Yi-Gan San (13) and BDW (14) formulation (Table 1). In the study with BDW (13), Sepia sp. and face powder were used as placebo; however, the way that the shellfish formulation was performed was not described. In the control group of the study using the Yi-Gan-San (14) formulation, 25 mg per day of tiapride hydrochloride were introduced. This drug is a substituted derivative with selective dopamine D2-receptor antagonist properties. This intervention (contamination) occurred in 44% of the individuals and was responsible for the symptoms, among them dizziness.
Generally, crude herbal drugs are natural products and their chemical composition depends on several factors such as geographic source of the plant material, climate in which it was grown, and time of harvest. Commercially available herbal medicinal products also vary in their content and concentration of chemical constituents from batch to batch and when products containing the same herbal ingredient are compared between manufacturers. Even when herbal products are standardized for content of known active or marker compounds to achieve more consistent pharmaceutical quality, variations in the concentrations of other constituents can be observed. The use of a protocol such as the Consolidated Standards of Reporting Trials (CONSORT), composed of 22 items, will probably minimize the limitations of RTC with phytotherapic agents (31).
The use of herbal medicines in the treatment of AD should be compared with the pharmacological treatment currently in use. Such studies should include the identification of the active principle in order to improve the validation of the clinical trial. Further large-scale, multicenter studies are necessary to determine the effectiveness of these substances in the cognitive deterioration of AD. Until then, this review provides some evidence of the benefit of Melissa, Salvia, Yi-Gan San and BDW in the treatment of AD.

herbs
alzheimer's disease
systematic review
dementia
elderly
cognitive impairment
randomized clinical trial


Integrative analysis of the mouse embryonic transcriptome

Monitoring global gene expression provides insight into how genes and regulatory signals work together to guide embryo development. The fields of developmental biology and teratology are now confronted with the need for automated access to a reference library of gene-expression signatures that benchmark programmed (genetic) and adaptive (environmental) regulation of the embryonic transcriptome. Such a library must be constructed from highly-distributed microarray data. Birth Defects Systems Manager (BDSM), an open access knowledge management system, provides custom software to mine public microarray data focused on developmental health and disease. The present study describes tools for seamless data integration in the BDSM library (MetaSample, MetaChip, CIAeasy) using the QueryBDSM module. A field test of the prototype was run using published microarray data series derived from a variety of laboratories, experiments, microarray platforms, organ systems, and developmental stages. The datasets focused on several developing systems in the mouse embryo, including preimplantation stages, heart and nerve development, testis and ovary development, and craniofacial development. Using BDSM data integration tools, a gene-expression signature for 346 genes was resolved that accurately classified samples by organ system and developmental sequence. The module builds a potential for the BDSM approach to decipher a large number developmental processes through comparative bioinformatics analysis of embryological systems at-risk for specific defects, using multiple scenarios to define the range of probabilities leading from molecular phenotype to clinical phenotype. We conclude that an integrative analysis of global gene-expression of the developing embryo can form the foundation for constructing a reference library of signaling pathways and networks for normal and abnormal regulation of the embryonic transcriptome. These tools are available free of charge from the web-site http://systemsanalysis.louisville.edu requiring only a short registration process.
Background
Animal development is fashioned by conserved signaling pathways that orchestrate morphogenesis, pattern formation, and cell differentiation - complex processes operating jointly in
different parts of an embryo and in stages associated with sequential gene activation. Monitoring local and temporal changes in gene expression can provide insight into how genes
and regulatory signals work together to guide development. [1] This knowledge is important for understanding the pathogenesis
of birth defects and to the central problems of defining precursor target cell susceptibility and the causal mechanisms of abnormal development triggered by diverse environmental and genetic
perturbations to maternal-fetal unit. [2]
Profiling gene expression on a global scale has become an important source of information for biological knowledge discovery. Despite well-known challenges confronting
technology development, the analysis of global gene expression data can reveal themes in the biologically robust response patterns in gene activity. [3] Gene Expression Omnibus (GEO)
is one of the main national repositories for high-information content transcript data from microarray analysis and serial analysis of gene expression (SAGE). [4] GEO has grown from
18,235 records in June 2004 to 115,415 records in December 2006, reflecting an average growth of over 100 new entries per day. A subset of this information describes the embryo proper
and can be mined for major biological themes in developmental health and disease. Using keyword searches and trend analysis to mine PubMed and Medline literature databases, the public
availability of embryo-based microarrays currently numbers 500-600 (564), mostly studies on mouse embryos and differentiating human cell lines.
The increasing volume of gene expression data on local and temporal states confronts the developmental biologist with the need for reference libraries and information management
systems to handle optimal-scale gene-expression signatures and facilitate biological knowledge discovery. For example, Lamb et al., [5] created a prototype reference collection of geneexpression
signatures from cultured human cells exposed to bioactive molecules, which serves as a platform for pattern-matching software to establish a ‘connectivity map’ between
drugs, genes, and diseases. Another example is the integrative analysis of multi-study tumor profiles. [6,7]
The emerging database model for tumor classification based on molecular abundance profiles has implied a 67-gene core ‘common transcriptional program’ in multiple cancers. [8] In developmentteratogenesis,
a preliminary meta-analysis across microarray studies in the mouse embryo returned a gene-expression signature of 512 developmentally regulated genes of which 16%
(~82-genes) changed during exposure to teratogenic agents. [2] Given the promises and pitfalls of computational methods for
solving gene expression problems, automated access to a reference collection of gene-expression signatures to benchmark the programmed (genetic) and adaptive (environmental)
regulation of the embryonic transcriptome is scientifically needed.
‘Birth Defects Systems Manager’ (BDSM) is a knowledge management system that provides custom software to mine public microarray data for interesting patterns across
developmental stages, organ systems, and disease phenotypes. [2,9]
This open resource enables: consolidation of communal data and metadata relevant to developmental health and disease; interactions with current builds of national databases and data
repositories; efficient algorithms for cross-species annotation of symbolic gene annotations using the NCBI sequence homologybased annotations for corresponding homologues and
orthologues; specific queries across experiments to facilitate secondary analysis; and data formats interoperable with analysis software for phenetic clustering, chromosomal mapping, gene
ontology classification, pathway evaluation, and network identification.
Since a comprehensive reference collection of gene-expression signatures for developing structures must be constructed from highly-distributed data, the present study was designed to
empower BDSM with tools for seamless data integration: MetaSample, MetaChip, and CIAeasy. These tools are accessible
at http://systemsanalysis.louisville.edu requiring only a short
registration process to BDSM. A field test of the prototype run with published microarray data illustrates proof of concept for integrative analysis of the mouse embryonic transcriptome.
Methodology
Dataset collections
Search of PubMed using keywords ‘embryo’ and ‘microarray’ returned 495 records of which 193 actually used the technology to study developing animal systems. GEO data sets (GDS) narrowed the list to 47 nonredundant
microarray datasets, and including the keyword ‘teratogen’ added a few more datasets, for a total of 564 public microarrays on the embryo. Raw and/or processed microarray
sample-data files and associated metadata were parsed onto the server using LoadBDSM. [9] BDSM currently holds 25
developmental series containing 537 samples that are derived from the public domain and 3 series containing 43 samples which are private. These data represent 15 developing organ
systems, 6 chemical exposures, and 5 drug interventions across 42 development stages.
Tracking provenance
For the present study, we restricted the analysis to 160 arrays in the BDSM library based on wellannotated experiments published on normal mouse
embryogenesis using an Affymetrix technology platform. These conditions are identified by GEO Series Accession number (GSE) and/or literature citation as follows: preimplantation
mouse embryo (GSE1749)  [10]; heart (GSE1479) GD10 - GD18 [11]; nerve (GSE972) GD9.5 - birth  [12];
ovary (GSE1359) and testis (GSE1358) between GD11.5 - birth  [13]; orofacial region
(GSE1624)  [14] and secondary palate  [15] between GD13-15.
The platforms for these series included: MG-U74Av2 (12488 probes), MG-U74Bv2 (12478 probes), MG430Av2 (45104 probes), MOE430Av2 (22690 probes), and MOE430Bv2 (22576
probes). Internal annotation of Affymetrix probe identifiers was performed to standardize gene labels across samples and improve cross-platform interoperability, as discussed previously.
[2]
Data integration
Individual microarray sample-data files from the aforementioned developmental series were integrated using QueryBDSM, a module that for merging pre-processed,
normalized samples from the BDSM library. Three metaanalysis tools written in PHP were designed to compare and analyze expression data across multiple chips and platforms:
MetaSample, MetaChip, and CIAeasy. The workflow schema is diagrammed in Figure 1. Individual sample-data files are
selected from the BDSM library and added to a queue for integration. The formatted input files are tab-delimited expression ratios of probes (rows) x samples (columns).
QueryBDSM determines the number of distinct microarray platforms in the sample queue and merges the data as follows: if all samples come from the same microarray platform (number
=1), then QueryBDSM automatically runs MetaSample to create a merged-table having ‘columns’ of normalized expression data (samples) in ‘rows’ derived from the platform, with unique probe
identifiers (ProbeID) expanded to include GenBank accession (GeneID), and symbolic gene name (Gene Symbol). If multiple platforms are represented by samples in the queue (number >1),
then QueryBDSM automatically runs MetaChip. MetaChip merges data when probe identifiers are different but represent the same annotation, such as across microarray platforms or
phylogenetic species. The probe identifiers from each platform are converted to UniGene ID and then merged accordingly, with associated expression data, for those genes common across the
datasets. The probes are annotated based on reverse-engineering the sequence homology-based annotations from GenBank. [2]
For this purpose the system uses data flat files downloaded monthly from the HomoloGene and UniGene databases of NCBI. [16]
In contrast to MetaSample and MetaChip, automated tools for merging samples, CIAeasy must be specified explicitly to
compare datasets for the same samples on different platforms. CIAeasy was created from the ADE-4 for R statistical
computing software package [17] and is adapted from ‘coinertia analysis’ (CIA) for microarrays. [18] With CIAeasy
users can perform CIA from the BDSM web site without detailed knowledge of R language programming. Since samples are aligned on a common space, CIA extracts
information about the joint trends in expression patterns of genes independent of probe or sequence annotation. [18]
CIAeasy automatically computes successive orthogonal axes with correspondence analysis and returns the percentage of total variance explained by each eigenvector to find the
strongest trends in the co-structured datasets.
Data analysis
One of the problems confronting meta-analysis is the normalcy and spread of expression data. In order to derive expression ratios from the Affymetrix data, we computed a
reference denominator, averaged for each gene at the earliest stage in a developmental series. These ratios were transformed to logarithm base 2 (log2) to produce a continuous spectrum of
values without biasing between up- and down-regulated genes and making the spread more normal. Each microarray set was centered to median of 0.00 and standardized by scaling to an
average standard deviation of 0.5. [2] The merged data were imported to GeneSpring v 7.2 using the UniGene cluster ID as
the unique gene identifier. The data were clustered using Pearson correlation for the gene tree and two-sided Spearman Confidence for the developmental conditions. Functional
annotation used the NIH/NIAID Database for Annotation, Visualization and Integrated Discovery (DAVID). [19] The
highest-ranking biological themes were stratified by Gene Ontology (GO) terms.
Discussion
Implementation of QueryBDSM
For proof of concept we examined samples from GEO data source GSE1391, a series describing global gene-expression profiles of the mouse embryo
during preimplantation stages.  [10] Samples included in this series represent development of the oocyte through fertilization
(1-cell embryo), activation of the zygotic genome (2-cell embryo) and first differentiation (8-cell embryo) leading to divergent embryonal (inner cell mass) and trophectodermal
(placental) lineages of the blastocyst. Biological replicates (3-4) arrayed at each stage used three different Affymetrix platforms: MOE430Av2 (22690 probes), MOE430Bv2 (22576 probes), and
MG-U74Av2 (12488 probes). Gene-expression profiles were normalized to the ‘oocyte’ in each platform as the earliest stage in the series. Derived data are log2-scale expression values
computed from the ratio of signals to the oocyte reference.
Using QueryBDSM, we merged datasets from the different samples to create three distinct datasets for the MOE430Av2, MOE430Bv2, and MG-U74Av2 platforms. Statistical
(ANOVA) analysis, run at high stringency with Benjamini-Hochberg correction applied, returned 4417 probes (alpha = 0.0001), 1614 probes (alpha = 0.0001), and 2400 probes (alpha
= 0.001) that were differentially regulated. Aside from 34 probes that overlapped between the first two datasets, different genes were detected across these diverse platforms. Hierarchical
clustering revealed two basic trajectories of gene-expression in all three platforms (Figure 2). One expression cluster contained genes that increased at the 2-cell stage to the blastocyst stage,
and the other cluster contained genes that decreased over these stages (not shown). These probes were mapped to the 307 reference pathways in the KEGG: Kyoto Encyclopedia of Genes
and Genomes (http://www.genome.ad.jp/kegg/) library to
identify metabolic themes. The top significant KEGG pathways showed concordance between platforms MOE430Av2 and MGU74Av2 (Table 1), whereas MOE430Bv2 detected different
pathways. Some pathways had marginal P-values with individual analysis that became significant in datasets joined by MetaChip (e.g., Adherens Junction, Tight Junction pathways).
This illustrates the strength of the meta-analysis approach.
We next used QueryBDSM to merge samples from platforms MOE430Av2 and MG-U74Av2 to illustrate the MetaChip and CIAeasy tools. These chips contained 13022 and 9562 nonredundant
UniGene cluster identifiers, respectively, of which 7278 were common between the two platforms when passed through MetaChip. Statistical (ANOVA) analysis with the same
parameters as before returned 3324 genes that were differentially regulated in this data subset. The top significant KEGG pathways for the combined 430A-U74A MetaChip are
concordant with the individual analysis (Table 1). CIAeasy was used to compare the joint trend between 4417, 1614, and 2400 probes from MetaSample, resulting in a high-level of similarity
between these three platforms (Figure 2). Of the 6812 nonredundant probes between MOE430Av2 and MG-U74Av2 (4417 + 2400), only five probes were common. Annotating the probes which were not in common gave 4551 unique DAVID
identifiers. Again, meta-analysis picked up most of the significant KEGG pathways identified in either of the singular analyses performed earlier and a few additional metabolic pathways (Table 1).
Comparative bioinformatics analysis across developing systems
An obvious limitation in combining data from several different platforms is that as more platforms are included, fewer representative genes are found to be common amongst all
platforms. This problem increases when considering less comprehensive arrays, older arrays with outdated probe annotations, or arrays across animal species. Staging the entry of
data from the most versatile arrays first can lessen the problem of losing information when data are combined across platforms; however, in some data mining efforts the discriminating power
gained by increasing samples and conditions might outweigh the loss of information. For example, multi-platform datasets have been found to discriminate tumor classification by expression
profile with as few as 25 genes. [6] For this reason it may be possible to benchmark developmental stages using a limited
number of genes across many diverse platforms. To illustrate this point we used BDSM-derived data to compare expression profiles across six unrelated studies and developing systems. We
constructed a virtual meta-chip for probes common to all five technology platforms represented in these studies, yielding 346 genes. Unsupervised clustering and Pearson correlation of the
gene-expression profiles correctly ordered the samples first by organ system and then by developmental sequence within each system (Figure 3). Within this hierarchy commonalities and
differences across organ systems were evident for the patterns of expression in subsets of genes. Unfortunately, the number of genes returned from 346 by statistical (ANOVA) analysis of
each individual system, or by K-means clustering of the entire matrix, was too small for insightful functional annotation. QueryBDSM is a simple and efficient solution that can be used
to construct a self-evolving reference collection of geneexpression profiles from highly-distributed data on mouse development. Although robust mapping of biological themes and pathways that are expressed at particular developmental
stages is straightforward when the same technology platform is considered  [2] fuzzy-clustering methods will be needed when multiple platforms are considered.
The MetaSample and MetaChip components of QueryBDSM are also available as standalone tools under the MetaBDSM module. In this way, the user can upload data outside the BDSM library.
The user supplies details of the technology platform, organism, and information about the file format. Once all the required fields are entered and submitted, the files are checked for unique
headers. Columns can be dropped by selecting the appropriate checkboxes. Clicking the Continue button combines the datasets with expression data and unique identifiers only for genes
common between the datasets. These tools have been tested on Internet Explorer 6.0 or greater and Mozilla-based browsers, such as Netscape 6.0 and Firefox.
Other tools are available to assemble data when the platform is same, such as Microarray data assembler. [20] This Excel-based
program inherits Excel's limitation from file sizes and number of samples (256 columns and 65,000 data points) whereas the MetaSample and MetaChip tools do not have this limitation.
These tools create temporary tables in the Oracle database and join them using the functionality of Oracle before putting it into a text file, reducing restrictions on the number of samples and
size of files. Although users can theoretically combine 100 files at a single time, it is not recommended to load more then 25 files at a time.
Conclusion
The representation of experimental samples as developmentally contiguous groups is expected to yield a novel mosaic view of gene-expression signatures and genetic dependencies. Although
sufficient data exists for data-mining efforts to begin, the ultimate goal of an unabridged reference collection must be viewed as a long-term effort. Regarding the embryo, a search of
OVID MedLine using keywords ‘embryo’ OR ‘teratogen’ (136,146 records) AND ‘microarray’ OR ‘SAGE’ (16,906 records) returned 343 total records. At the current rate of 564
microarrays per 343 publication records (factor = 1.64), the trajectory of embryo-based microarray publications projects GEO to hold in excess of 1,476 microarrays relevant for
embryogenesis or teratogenesis by the year 2010.
As studies unravel gene-expression signatures, the key principles in teratology – namely, chemical effects on biological mechanisms, dose-response relationships, factors underlying
genetic susceptibility, stage-dependent responses, and maternal influences, can be framed in a systems biology context to address an ‘experience database’ for ranking pathways and
networks by strength of association with anatomical landmarks and developmental abnormalities.  [2] The BDSM resource
would parallel efforts toward molecular diagnostics in cancer biology (http://www.oncomine.org/), which includes data sets
profiling human tumor samples. [21] Since interpreting geneexpression signatures in birth defects will be predicated on
posterior (prior) knowledge about developmental health and disease, an important payoff from this bioinformatics effort is to recognize and characterize how these biological states emerge
from adaptation or adverse regulation of the embryonic transcriptome.

integrative analysis
mouse
embryo
transcriptome
expression
birth defects