Differences In Cellular Composition Research Articles

Correcting for cell-type composition bias in epigenome-wide association studies.

Recent epigenome-wide association studies have indicated a potential role for epigenetic variation in the etiology of complex human diseases. However, one major challenge is to distinguish true epigenetic variation from changes caused by differences in cellular composition between the disease and non-disease state, a problem that is particularly relevant when analyzing whole blood. For studies with large numbers of samples, it can be expensive and very time consuming to perform cell sorting, and it is often not clear which is the correct cell type to profile. Two recently published papers have attempted to address this confounding issue using bioinformatics.

Epigenome-wide differences in pathology-free regions of multiple sclerosis-affected brains.

Using the Illumina 450K array and a stringent statistical analysis with age and gender correction, we report genome-wide differences in DNA methylation between pathology-free regions derived from human multiple sclerosis-affected and control brains. Differences were subtle, but widespread and reproducible in an independent validation cohort. The transcriptional consequences of differential DNA methylation were further defined by genome-wide RNA-sequencing analysis and validated in two independent cohorts. Genes regulating oligodendrocyte survival, such as BCL2L2 and NDRG1, were hypermethylated and expressed at lower levels in multiple sclerosis-affected brains than in controls, while genes related to proteolytic processing (for example, LGMN, CTSZ) were hypomethylated and expressed at higher levels. These results were not due to differences in cellular composition between multiple sclerosis and controls. Thus, epigenomic changes in genes affecting oligodendrocyte susceptibility to damage are detected in pathology-free areas of multiple sclerosis-affected brains.

High-fat diet alters gut microbiota physiology in mice

The intestinal microbiota is known to regulate host energy homeostasis and can be influenced by high-calorie diets. However, changes affecting the ecosystem at the functional level are still not well characterized. We measured shifts in cecal bacterial communities in mice fed a carbohydrate or high-fat (HF) diet for 12 weeks at the level of the following: (i) diversity and taxa distribution by high-throughput 16S ribosomal RNA gene sequencing; (ii) bulk and single-cell chemical composition by Fourier-transform infrared- (FT-IR) and Raman micro-spectroscopy and (iii) metaproteome and metabolome via high-resolution mass spectrometry. High-fat diet caused shifts in the diversity of dominant gut bacteria and altered the proportion of Ruminococcaceae (decrease) and Rikenellaceae (increase). FT-IR spectroscopy revealed that the impact of the diet on cecal chemical fingerprints is greater than the impact of microbiota composition. Diet-driven changes in biochemical fingerprints of members of the Bacteroidales and Lachnospiraceae were also observed at the level of single cells, indicating that there were distinct differences in cellular composition of dominant phylotypes under different diets. Metaproteome and metabolome analyses based on the occurrence of 1760 bacterial proteins and 86 annotated metabolites revealed distinct HF diet-specific profiles. Alteration of hormonal and anti-microbial networks, bile acid and bilirubin metabolism and shifts towards amino acid and simple sugars metabolism were observed. We conclude that a HF diet markedly affects the gut bacterial ecosystem at the functional level.

Translating translational research: mouse models of human disease

Translating translational research: mouse models of human disease

Abstract 1818: Gene expression profiling to understand stages in breast cancer development and progression: importance of tissue sampling and context .

Abstract Background: During the past decade, gene expression profiling has been widely employed to understand the heterogeneity of tumor biology. Novel biomarkers and signatures discovered using these techniques have impacted tumor diagnosis and prognosis, and have led to important insights into etiology. However, application of mRNA profiling to define mechanisms mediating breast cancer risk factor exposure remains underexplored. Identifying mediating pathways of breast carcinogenesis by tumor tissue profiling may be problematic because genomic instability may obscure causal changes. Methods: To evaluate how etiologically relevant signatures are expressed during different stages of carcinogenesis, we analyzed gene expression data from three types of whole tissue samples: benign tissues from non-diseased breasts (“normal”), benign tissues adjacent to cancer (“cancer-associated”), and tumors. We assessed three published signatures (Age, Obesity, and Parity, originally defined in “normal” breast tissues from healthy women) in 149 tumor tissues and paired cancer-adjacent benign tissues from the Polish Women's Breast Cancer Study. Associations of chronological age, body mass index and parity with their related mRNA signatures were evaluated. Results: The association between Obesity signature and actual obesity status showed a marked gradient from normal breast tissues (obese vs. non-obese: OR=5.78, p=0.0009) to cancer-adjacent and tumor tissues (cancer-adjacent: OR=1.85, p=0.09; tumor: OR=0.80, p=0.54). A similar pattern was also observed for Age signature (normal: OR [<40 y vs. ≥ 40y] =5.03, p<0.0001; cancer-adjacent: OR [<45 y vs. ≥ 45y] =6.97, p=0.005; tumor: OR=0.58 p=0.35) and Parity signature (parous vs. nulliparous, normal: OR =3.95, p=0.0002; cancer-adjacent: OR=1.38, p=0.44; tumor: OR=1.20, p=0.65). Simulation studies further demonstrated that the association strength was influenced by the proportion of risk factor-associated genes that were dysregulated during carcinogenesis. Differences in cellular composition between samples may also affect association strength. Conclusions: These results suggest that associations between risk factors and gene signatures are strongest in benign tissues from non-diseased breasts and may be weakened by the responses of cancer-adjacent issues to cancer progression, and by the effects of genomic instability in tumors. While tumor tissue is readily available in many epidemiologic studies, use of this tissue for studying etiologically relevant pathways may have important limitations. Identification of etiologically relevant pathways may rely on studies of well-annotated tissues from women without cancer. Citation Format: Xuezheng Sun, Alexandre Lockhart, Gretchen L. Gierach, Mark Sherman, Jonine D. Figueroa, Sallie S. Schneider, D. Joseph Jerry, Melissa A. Troester. Gene expression profiling to understand stages in breast cancer development and progression: importance of tissue sampling and context . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1818. doi:10.1158/1538-7445.AM2013-1818 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Analysis of Draining Leukocytes from the Abdominal Fluid to Monitor Immune Events after Intestinal Transplantation. Follow Up Report

Introduction: During intestinal transplant (ITx) operation, intestinal lymphatics are not reconstituted. Consequently, lymph containing trafficking leukocytes drains freely into the abdominal cavity, where this fluid is absorbed by an open drainage system. We have previously reported that the abdominal drainage sample is different from the blood sample in composition of the three major leukocyte populations, lymphocytes, monocytes, and granulocytes and therefore might be useful for monitoring immune events during the first post-operative weeks. In this follow up study, our aim was to correlate changes in fluid cellularity with the occurrence of clinical events. Methods: Seventeen consecutive ITx patients (12 pediatric, 5 adult; 1 liver/intestine, 2 multivisceral, 14 isolated) were included in this retrospective cohort study. Cell composition of the abdominal drainage sample was analyzed by flow cytometry during the first 15 post-ITx days. The correlation between cell parameters (FSC-SSC pattern, CD4/CD8 ratio, CD69 expression in T cells, NK and NKT cells appearance) and clinical complications was evaluated. Results: Cellular pattern varied along the post-ITx period in non-complicated recipients (n = 6) from a mixed leukocyte pattern to an exclusively lymphocytic pattern from the fourth day onwards with predominance of CD4+ T cells (50-80%). In contrary, in samples of patients with intra-abdominal clinical complications, such as bacterial infections (n = 3), rejection (n = 3), or non-infectious inflammatory events (n = 5), we could correlate appearance of granulocytes and natural killer cells with clinical events. In addition, the expression of the early activation marker CD69 was significantly increased on CD4 and CD8 T cells from samples with clinical events. No clear differences in cellular composition or CD69 expression between the different clinical complications could be detected. Conclusions: Our results indicate that cell analysis of the draining fluid from intestinal transplant recipients might be useful as a tool to support diagnosis and clinical patient management during the first post-Tx weeks, that are critical due to high incidence of complications. Furthermore, this follow-up study ensures the utility of this method to gain insight into intestinal transplant immunobiology.

Cellular responses of the long-spined sea urchin (Diadema antillarum-a)(160.9)

Abstract The long-spined sea urchin (Diadema antillarum-a) began dying on coral reefs near the Panama Canal in 1983, and over the next year they virtually disappeared from the Caribbean Sea. Present evidence suggests that a water-borne pathogen was responsible. Since their die-off Diadema densities remain low, and as they facilitate coral recruitment and persistence by controlling macroalgae, its loss has contributed to the transformation of living coral reefs in the Caribbean to barren algae-covered rock. Our preliminary on-site studies of the humoral responses of the urchins showed that Diadema coelomocytes did not respond to lipopolysaccharide as vigorously as did other common Caribbean sea urchins (Tripneustes ventricosa, Echinometra lucunter, and E. viridis). We then studied the cellular immune responses of coelomocytes and coelomic fluid of Diadema and those of the other urchins unaffected by die-off. Light microscopy revealed differences in coelomocyte populations of the predominant urchins of the Caribbean. We used flow cytometry to investigate differences in cellular composition and responses of coelomocytes in phagocytosis assays. It is hoped that this research will increase insight into one of the most devastating and ecologically important die-offs ever recorded, as well as provide the information required to understand whether weakened immunity was involved in the mass mortality of Diadema antillarum-a, and whether strengthening of immunity has occurred since then.

FOXP3+ regulatory and TIA‐1+ cytotoxic T lymphocytes in HIV‐associated Hodgkin lymphoma

Human immunodeficiency virus (HIV) infects CD4(+) lymphocytes, leading to a development of malignant lymphomas, such as HIV-associated Hodgkin Lymphoma (HIV-HL). This study aimed to assess the differences in cellular composition of the inflammatory reactive background of HIV-HLs. We examined infiltrating T lymphocytes, specifically regulatory T cells, cytotoxic cells, Epstein-Barr virus (EBV) related antigens and HIV-receptor CCR5. In all HIV-HL cases, Hodgkin and Reed-Sternberg (HRS) cells showed EBER1 expression, LMP-1 staining positivity and EBNA-2 staining negativity, except for one case which showed LMP-1 staining negativity. Our histological findings indicate the percentage of CD8(+) , TIA-1(+) lymphocytes was significantly higher in HIV-HL than in non-HIV-HL cases (P < 0.05). On the other hand, the percentage of CD4(+) , FOXP3(+) lymphocytes was significantly lower in HIV-HL than in non-HIV-HL cases (P < 0.05) but present. The percentage of CCR5(+) lymphocytes was significantly lower in HIV-HL than in non-HIV-HL cases (P < 0.05). Usually, CD4(+) and CCR5(+) lymphocytes are reported to be rarely detected in HIV-associated non-Hodgkin lymphomas, but the presence of CD4(+) and/or FOXP3(+) lymphocytes may be implicated in the pathogenesis of HL. In addition, although additional CD8(+) lymphocytes are probably not EBV-LMP specific cytotoxic T-cells, these lymphocytes may also well be involved in the pathogenesis of HIV-HL.

Deletion of L-Selectin Increases Atherosclerosis Development in ApoE−/− Mice

Atherosclerosis is an inflammatory disease characterized by accumulation of leukocytes in the arterial intima. Members of the selectin family of adhesion molecules are important mediators of leukocyte extravasation. However, it is unclear whether L-selectin (L-sel) is involved in the pathogenesis of atherosclerosis. In the present study, mice deficient in L-selectin (L-sel −/−) animals were crossed with mice lacking Apolipoprotein E (ApoE −/−). The development of atherosclerosis was analyzed in double-knockout ApoE/L-sel (ApoE −/− L-sel −/−) mice and the corresponding ApoE −/− controls fed either a normal or a high cholesterol diet (HCD). After 6 weeks of HCD, aortic lesions were increased two-fold in ApoE −/− L-sel −/− mice as compared to ApoE −/− controls (2.46%±0.54% vs 1.28%±0.24% of total aortic area; p<0.05). Formation of atherosclerotic lesions was also enhanced in 6-month-old ApoE −/− L-sel −/− animals fed a normal diet (10.45%±2.58% vs 1.87%±0.37%; p<0.05). In contrast, after 12 weeks of HCD, there was no difference in atheroma formation between ApoE −/− L-sel −/− and ApoE −/− mice. Serum cholesterol levels remained unchanged by L-sel deletion. Atherosclerotic plaques did not exhibit any differences in cellular composition assessed by immunohistochemistry for CD68, CD3, CD4, and CD8 in ApoE −/− L-sel −/− as compared to ApoE −/− mice. Leukocyte rolling on lesions in the aorta was similar in ApoE −/− L-sel −/− and ApoE −/− animals. ApoE −/− L-sel −/− mice exhibited reduced size and cellularity of peripheral lymph nodes, increased size of spleen, and increased number of peripheral lymphocytes as compared to ApoE −/− controls. These data indicate that L-sel does not promote atherosclerotic lesion formation and suggest that it rather protects from early atherosclerosis.

The Calreticulin-Binding Sequence of Thrombospondin 1 Regulates Collagen Expression and Organization During Tissue Remodeling

Amino acids 17-35 of the thrombospondin1 (TSP1) N-terminal domain (NTD) bind cell surface calreticulin to signal focal adhesion disassembly, cell migration, and anoikis resistance in vitro. However, the in vivo relevance of this signaling pathway has not been previously determined. We engineered local in vivo expression of the TSP1 calreticulin-binding sequence to determine the role of TSP1 in tissue remodeling. Surgical sponges impregnated with a plasmid encoding the secreted calreticulin-binding sequence [NTD (1-35)-EGFP] or a control sequence [mod NTD (1-35)-EGFP] tagged with enhanced green fluorescent protein were implanted subcutaneously in mice. Sponges expressing NTD (1-35)-EFGP formed a highly organized capsule despite no differences in cellular composition, suggesting stimulation of collagen deposition by the calreticulin-binding sequence of TSP1. TSP1, recombinant NTD, or a peptide of the TSP1 calreticulin-binding sequence (hep I) increased both collagen expression and matrix deposition by fibroblasts in vitro. TSP1 stimulation of collagen was inhibited by a peptide that blocks TSP1 binding to calreticulin, demonstrating the requirement for cell surface calreticulin. Collagen stimulation was independent of TGF-β activity and Smad phosphorylation but was blocked by an Akt inhibitor, suggesting that signaling through the Akt pathway is important for regulation of collagen through TSP1 binding to calreticulin. These studies identify a novel function for the NTD of TSP1 as a mediator of collagen expression and deposition during tissue remodeling.

Maintenance of regional difference in cellular composition of neurospheres derived from adult mouse olfactory bulb

Neurospheres are potentially good tools to study olfactory neurogenesis. For this purpose, it is necessary to characterize neurospheres derived from the olfactory system. This study is aimed at identifying the regional difference of cellular composition within neurospheres derived from the adult mouse olfactory bulb, and studying whether these characteristics are maintained throughout the long-term culture. Neural cells were obtained from the olfactory bulbs of 8-week-old Balb/c mice and the dissociated cells were cultured in serum-free media containing epidermal growth factor and basic fibroblast growth factor to form neurospheres. These neurospheres were subjected to characterization by confocal microscopy after immunofluorescence staining with nestin, proliferating cell nuclear antigen (PCNA), neuronal cell adhesion molecule (NCAM), glial fibrillary acidic protein (GFAP), O4, and olfactory marker protein (OMP). Confocal microscopy showed that nestin-reactive cells, which are stem cell-like cells, were present at the periphery of neurospheres and PCNA-reactive cells undergoing proliferation were found throughout the neurospheres. Neuronal cell markers, NCAM-reactive cells, were observed at the center of neurospheres while glial cell markers, GFAP- or O4-reactive cells, were present at the periphery of neurospheres. No cells were found to be reactive for OMP, a differentiated olfactory neuron marker. This regional distribution of cells in neurospheres was maintained through 17 passages for a year. The neural lineage of cells showed different distribution within neurospheres and this localization was preserved throughout the culture period. These findings in the neurosphere culture system of the olfactory bulb may help to study olfactory neurogenesis.

Immune reconstitution after autologous hematopoietic transplantation with Lin −, CD34 +, Thy-1 lo selected or intact stem cell products

In sequential studies, we compared immune reconstitution following high-dose chemotherapy (HDT) and stem cell transplantation (SCT) using intact mobilized peripheral blood stem cell (PSC) in intermediate grade non-Hodgkin's lymphoma (NHL) patients and CD34 +, lineage-negative (Lin −), Thy-1 lo (CD34 +Lin −Thy-1 lo) stem cells in low-grade NHL patients. Cytokine expression and cellular phenotype and function were used as the basis of comparison. Despite differences in cellular composition of the stem cell grafts, immune reconstitution in both groups was similar. Significantly higher levels of type 1- and 2-associated cytokine messenger ribonucleic acid (mRNA) were observed both prior to and following transplant in the peripheral blood (PB) of both cohorts as compared to normal individuals. Similar levels of interleukin (IL)-4, IL-10, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) messenger ribonucleic acid (mRNA) were seen in PB mononuclear cells following transplant with either product. In contrast, patients receiving isolated CD34 +Lin −Thy-1 lo cells expressed significantly higher IL-2 levels at all times examined post-transplant. Despite the high levels of cytokine gene expression and rapid restoration to pretransplant levels of CD3 cell number by day 30, T cell function and CD4:CD8 and CD4 +CD45RA:CD4 +CD45RO + ratios were significantly depressed in both cohorts compared to normal donors, and significantly lower in patients transplanted with CD34 +Lin −Thy-1 lo compared to patients receiving an intact PSC product. These data suggest that the peripheral tolerance in patients receiving HDT and an autologous SCT occurs independent of graft composition, although immune function and CD4 recovery are better facilitated by transplantation of an intact product.

Transcriptional responses in mouse lungs induced by vaccination with Mycobacterium bovis BCG and infection with Mycobacterium tuberculosis

Transcriptome analyses enable the assessment of signature alterations in whole tissues and organs undergoing pathological processes. We analyzed gene expression profiles of lungs from mice infected with Mycobacterium tuberculosis or vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG). We compared high-dose systemic and low-dose aerosol M. tuberculosis infections as well as systemic BCG vaccination. Expression profiles in lungs were analyzed at day (d) 1 and d 30 post infection / vaccination using a custom tailored 'in situ' synthesized 60-mer oligonucleotide microarray with focus on immunologically relevant genes. At d 1, a small number of genes were differentially regulated, whereas at d 30, a discrete expression pattern was identified in the lung. Differential gene expression profiles between M. tuberculosis infection and BCG vaccination indicate differences in naturally and vaccine induced pulmonary immune responses. The shared signature of systemic and aerosol M. tuberculosis infection revealed dominance of genes related to or controlled by interferon gamma (IFN-γ). We assume that differential gene expression profiles after M. tuberculosis infection are strongly influenced by differences in cellular composition of the lung due to migration of immune cells, primarily neutrophils, basophils, eosinophils and monocytes to the site of infection.

Experimental trial for diagnosis of pancreatic ductal carcinoma based on gene expression profiles of pancreatic ductal cells

Pancreatic ductal carcinoma (PDC) remains one of the most intractable human malignancies, mainly because of the lack of sensitive detection methods. Although gene expression profiling by DNA microarray analysis is a promising tool for the development of such detection systems, a simple comparison of pancreatic tissues may yield misleading data that reflect only differences in cellular composition. To directly compare PDC cells with normal pancreatic ductal cells, we purified MUC1-positive epithelial cells from the pancreatic juices of 25 individuals with a normal pancreas and 24 patients with PDC. The gene expression profiles of these 49 specimens were determined with DNA microarrays containing >44 000 probe sets. Application of both Welch's analysis of variance and effect size-based selection to the expression data resulted in the identification of 21 probe sets corresponding to 20 genes whose expression was highly associated with clinical diagnosis. Furthermore, correspondence analysis and 3-D projection with these probe sets resulted in separation of the transcriptomes of pancreatic ductal cells into distinct but overlapping spaces corresponding to the two clinical classes. To establish an accurate transcriptome-based diagnosis system for PDC, we applied supervised class prediction algorithms to our large data set. With the expression profiles of only five predictor genes, the weighted vote method diagnosed the class of samples with an accuracy of 81.6%. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC.

Quantitative proton MR spectroscopic imaging of the mesial temporal lobe

To evaluate variations in regional metabolite concentrations in the anterior mesial temporal lobe (ATL), and compare metabolite concentrations between the allocortex and neocortex using quantitative proton MR spectroscopic imaging (MRSI). Metabolite concentrations and ratios were measured in 20 healthy young subjects with the use of a multislice spin-echo (SE) sequence (TR/TE=2300/280 msec). Quantitation of MRSI data was performed by means of the phantom replacement methodology. The highest choline (Cho) concentration (4.1 +/- 1.1 mM) was found in the ATL (P=0.0015 compared to the middle mesial temporal lobe (MTL), and P=0.0008 compared to the posterior mesial temporal lobe (PTL)). The ATL also had a higher Cho/creatine (Cr) ratio and a lower N-acetyl aspartate (NAA)/Cho ratio compared to other examined regions (P <0.0001 and P < or = 0.052, respectively). In the allocortical regions, the average Cho concentration (3.5 +/- 0.8 mM) was 68% higher, and the NAA concentration (9.5 +/- 1.8 mM) was 13% lower than in the neocortex (P <10(-6) and P <0.008, respectively). Cho/Cr was 64% higher, NAA/Cr 14% lower, and NAA/Cho 47% lower in the allocortex than in the neocortex (P <10(-6), P=0.013, and P <10(-6), respectively). The mesial temporal lobe shows high levels of Cho, which presumably reflect a difference in cellular composition between the allocortex and neocortex. Regional metabolite variations must be considered when pathological conditions involving the mesial temporal lobe are evaluated.

The Role of Excitotoxicity in Secondary Mechanisms of Spinal Cord Injury: A Review with an Emphasis on the Implications for White Matter Degeneration

Following an initial impact after spinal cord injury (SCI), there is a cascade of downstream events termed 'secondary injury', which culminate in progressive degenerative events in the spinal cord. These secondary injury mechanisms include, but are not limited to, ischemia, inflammation, free radical-induced cell death, glutamate excitotoxicity, cytoskeletal degradation and induction of extrinsic and intrinsic apoptotic pathways. There is emerging evidence that glutamate excitotoxicity plays a key role not only in neuronal cell death but also in delayed posttraumatic spinal cord white matter degeneration. Importantly however, the differences in cellular composition and expression of specific types of glutamate receptors in grey versus white matter require a compartmentalized approach to understand the mechanisms of secondary injury after SCI. This review examines mechanisms of secondary white matter injury with particular emphasis on glutamate excitotoxicity and the potential link of this mechanism to apoptosis. Recent studies have provided new insights into the mechanisms of glutamate release and its potential targets, as well as the downstream pathways associated with glutamate receptor activation in specific types of cells. Evidence from molecular and functional expression of glutamatergic AMPA receptors in white matter glia (and possibly axons), the protective effects of AMPA/kainate antagonists in posttraumatic white matter axonal function, and the vulnerability of oligodendrocytes to excitotoxic cell death suggest that glutamate excitotoxicity is associated with oligodendrocyte apoptosis. The latter mechanism appears key to glutamatergic white matter degeneration after SCI and may represent an attractive therapeutic target.

The influence of changes in nitrogen: silicon ratios on diatom growth dynamics

Nitrate loading to coastal waters has increased over recent decades while silicon loading has remained relatively constant or decreased. As the N:Si ratio in coastal waters shifts due to these anthropogenic influences, silicate limitation of diatom biomass may become a feature of the biogeochemistry in coastal waters especially in regions of reduced exchange. Two sets of nutrient enrichment mesocosm experiments were conducted in successive years using a natural planktonic assemblage obtained from the Trondheimsfjord, Norway. The inorganic nutrient concentrations at the start of the experiments were manipulated to give a variety of N:Si concentrations at ratios representative of current and possible future values, should N loading continue. In June 1999 experiments were conducted with a gradient of inorganic N:Si ratios (1:2, 1:1, 2:1, 4:1) to investigate the influence of low and high N:Si ratio conditions and to determine the conditions that would generate Si limitation of diatom growth. In June 2000, based on 1999 data, highly replicated experiments were conducted at N:Si ratios of 1:1 and 4:1 which were expected to result in N and Si limitation of diatom growth, respectively; statistical differences in cellular composition were recorded. N limitation of diatom biomass increase was observed under the three lowest N:Si ratios: particulate carbon (C) accumulation continued to occur following N exhaustion resulting in an increase in the organic C:N ratio. Silicate limitation of diatom biomass increase only occurred at the highest N:Si ratio of 4:1. Silicate exhaustion was followed by continued nitrate uptake for several days, at a slower rate than previously. The resulting increase in organic N was accompanied by an increase in organic C such that the C:N ratio of the organic material at the highest N:Si ratio failed to increase to the extent observed under the N limited conditions. Statistically significant differences in chlorophyll-a yield per unit nitrate, C:chlorophyll-a ratios, C:N ratio and diatom cell yield per unit nitrate or Si were observed in Si compared to N limited conditions. All mesocosms became dominated numerically and in terms of biomass by the diatom Skeletonema costatum. The potential implications of changing N and Si regimes in coastal waters are discussed.

Immunohistochemical detection of pituitary transcription factor‐1 in paraffin sections of bovine adenohypophysis after wet heat‐induced antigen retrieval

AbstractPituitary transcription factor‐1 (Pit‐1) is a tissue‐specific transcription factor responsible for somatotroph (GH cell) development and growth hormone (GH) gene activation. To investigate the expression of Pit‐1 protein in bovine adenohypophysis, antigen retrieval conditions for detection of Pit‐1 were optimized in paraffin sections of bovine pituitary. We then localized Pit‐1 and GH employing a double‐immunohistochemical staining method and compared the distribution and proportion of adenohypophyseal cells containing Pit‐1 and GH between Japanese Black (Blk) and Holstein (Hol) steers. Autoclave heating pretreatment of sections in Dako Target Retrieval Solution for 20–30 min enabled reliable detection of the distinct nuclear immunostaining of Pit‐1. However, heat treatment in phosphate buffered saline or citrate buffer gave only a weak signal with increased background staining and false positive immunoreactions in the cytoplasm. Pituitary transcription factor‐1 was abundantly expressed in the bovine anterior pituitary and the number of Pit‐1 immunoreactive cells did not differ between the breeds. The proportion of GH cells relative to Pit‐1 immunoreactive cells was smaller in Blk than Hol steers, suggesting an intrinsic breed difference in cellular composition of bovine adenohypophysis. The immunohistochemical technique described will facilitate further investigation of the role played by Pit‐1 in the regulation of GH cell development and function in cattle.

Hybrid Carcinomas of the Salivary Glands: Report of Nine Cases with a Clinicopathologic, Immunohistochemical, and p53 Gene Alteration Analysis

Hybrid carcinomas of the salivary gland are a recently defined and rare tumor entity, consisting of two histologically distinct types of carcinoma within the same topographic area. In this study, we examined nine such cases, which mainly arose in the parotid gland (seven cases), with an additional one each from submandibular and lacrimal glands, and analyzed their clinicopathologic profiles, including immunohistochemical features and p53 gene alterations. The prevalence of hybrid carcinomas was 0.4% among the 1863 cases of parotid gland tumors in our series. The nine patients comprised five men and four women, ranging in age from 40 to 81 years (mean, 62 y). Tumor size ranged from 2 to 10 cm (mean, 4.2 cm). Of the seven patients who were followed up, two were alive with disease and five were alive with no evidence of disease, although the follow-up period was short. Three cases had cervical lymph nodal metastases. The combinations of carcinoma components in our hybrid carcinomas were as follows: epithelial–myoepithelial carcinoma and basal cell adenocarcinoma in two cases, epithelial–myoepithelial carcinoma and squamous cell carcinoma in one case, salivary duct carcinoma and adenoid cystic carcinoma in two cases, myoepithelial carcinoma and salivary duct carcinoma in one, acinic cell carcinoma and salivary duct carcinoma in one, and squamous cell carcinoma and salivary duct carcinoma in two. Although the proportion of each carcinoma component in a tumor mass varied from case to case, the minor component always represented ≥ 10% of the area. Differences in cellular composition were studied by immunohistochemistry and electron microscopy. The Ki-67–labeling index apparently differed between the two carcinoma elements in five cases. Diffusely positive p53 immunoreactivity was observed in four cases, restricted to the more aggressive component in each pair. Furthermore, p53 gene alteration analysis of these p53-positive cases revealed that all and three cases demonstrated loss of heterozygosity at p53 microsatellite loci and p53 gene point mutations, respectively, which were detected only in the p53-immunoreactive carcinoma component. Therefore, there is the possibility that such molecular-genetic events take an integral part for inducing the transformation from histologically lower to higher grade tumor during the hybrid carcinoma genesis of the salivary glands.

Distinguishing subtypes of temporal lobe epilepsy with background hippocampal activity.

Two subtypes of temporal lobe epilepsy (TLE) can be defined through clinical observations and analysis of hippocampal tissue resected during surgical procedures for intractable TLE: (a) mesial temporal sclerosis (MTS), which is characterized by extensive changes to the hippocampus and good surgical outcome; and (b) paradoxical temporal lobe epilepsy (PTLE), which is characterized by minimal cell loss and comparatively poorer surgical outcome. Patients in both subtypes have seizures that appear to begin in the medial temporal lobe, but documented differences in substrate and outcome between these subtypes has defined a need to distinguish MTS and PTLE patients before surgery. This report describes a retrospective study to investigate the feasibility of doing so during intracranial monitoring. Background EEG epochs, 5 min in duration, were recorded from the anterior hippocampus in 14 (10 MTS and four PTLE) patients with consistent localization of seizure onset to medial temporal structures. The power spectral density (PSD) of the EEG epochs was calculated by a Fourier spectral estimator, and the total signal power and power of the delta, theta, alpha, beta, and gamma frequency bands were submitted to group-to-group comparison. Spectral peaks were observed in the delta band in all PSD estimates and in the theta band in nine of 14 (seven MTS, two PTLE) estimates. The MTS and PTLE subtypes could be distinguished by the total signal power and delta band power. These power measurements were greater in the PTLE subtype. Both delta and theta spectral components are present in hippocampal background EEGs recorded from patients with TLE. The results indicate that group differences exist in spectral measures of background hippocampal signals recorded from MTS and PTLE subtypes. This suggests both that substrate differences in cellular composition and connectivity are reflected in hippocampal background EEGs and that spectral measurements of these signals may hold promise for tests to identify the group membership of individual patients.