FHOD1 and FMNL1 formin proteins in intestinal gastric cancer: correlation with tumor-infiltrating T lymphocytes and molecular subtypes.

BACKGROUND: Gastric cancer (GC) is the third most common cause of cancer death. Intestinal type GC is a molecularly diverse disease. Formins control cytoskeletal processes and have been implicated in the progression of many cancers. Their clinical significance in GC remains unclear. Here, we characterize the expression of formin proteins FHOD1 and FMNL1 in intestinal GC tissue samples and investigate their association with clinical parameters, GC molecular subtypes and intratumoral T lymphocytes. METHODS: The prognostic significance of FHOD1 and FMNL1 mRNA expression was studied with Kaplan-Meier analyses in an online database. The expression of FHOD1 and FMNL1 proteins was characterized in GC cells, and in non-neoplastic and malignant tissues utilizing tumor microarrays of intestinal GC representing different molecular subtypes. FHOD1 and FMNL1 expression was correlated with clinical parameters, molecular features and T lymphocyte infiltration. Immunohistochemical expression of neither formin correlated with survival. RESULTS: Kaplan-Meier analysis associated high FHOD1 and FMNL1 mRNA expression with reduced overall survival (OS). Characterization of FHOD1 and FMNL1 in GC cells showed cytoplasmic expression along the actin filaments. Similar pattern was recapitulated in GC tissue samples. Elevated FMNL1 was associated with larger tumor size and higher disease stage. Downregulation of FHOD1 associated with TP53-mutated GC tumors. Tumor cell FHOD1 expression strongly correlated with high numbers of tumor-infiltrating CD8 + lymphocytes. CONCLUSIONS: FHOD1 and FMNL1 proteins are expressed in the tumor cells of intestinal GC and significantly associate with clinical parameters without direct prognostic significance. FHOD1 correlates with high intratumoral CD8 + T lymphocyte infiltration in this cohort.

EGFR gene copy number predicts response to anti-EGFR treatment in RAS wild type and RAS/BRAF/PIK3CA wild type metastatic colorectal cancer.

Anti-EGFR antibodies are used for the treatment of RAS wild type metastatic colorectal cancer. We previously showed that EGFR gene copy number (GCN) predicts response to anti-EGFR therapy in KRAS exon 2 wild type metastatic colorectal cancer. The aim of our study was to analyse the predictive role of EGFR GCN in RAS/BRAF/PIK3CA wild type metastatic colorectal cancer. The material included 102 patients with KRAS exon 2 wild type metastatic colorectal cancer treated with anti-EGFR ± cytotoxic therapy. Next generation sequencing was used for KRAS, NRAS, BRAF and PIK3CA gene mutation analyses. EGFR GCN was analysed by EGFR immunohistochemistry guided automated silver in situ hybridisation. Increased EGFR GCN (≥4.0) predicted a better response and prolonged progression free survival in anti-EGFR treated RAS/BRAF/PIK3CA wild type patients (Log-rank test, p = 0.0004). In contrast, survival of RAS/BRAF/PIK3CA wild type, EGFR GCN below 4.0 patients did not differ from patients with mutant RAS, BRAF or PIK3CA. Our study indicates that EGFR GCN predicts anti-EGFR treatment efficacy in patients with RAS/BRAF/PIK3CA wt metastatic CRC. Tumours with EGFR GCN below 4.0 appear to be as refractory to anti-EGFR treatment as tumours with mutation in any of the RAS/RAF/PIK3CA pathway genes.

PTTG1-interacting protein (PTTG1IP/PBF) predicts breast cancer survival.

BACKGROUND: PTTG1-interacting protein (PTTG1IP) is an oncogenic protein, which participates in metaphase-anaphase transition of the cell cycle through activation of securin (PTTG1). PTTG1IP promotes the shift of securin from the cell cytoplasm to the nucleus, allowing the interaction between separase and securin. PTTG1IP overexpression has been previously observed in malignant disease, e.g. in breast carcinoma. However, the prognostic value of PTTG1IP in breast carcinoma patients has not previously been revealed. METHODS: A total of 497 breast carcinoma patients with up to 22-year follow-up were analysed for PTTG1IP and securin immunoexpression. The results were evaluated for correlations with the clinical prognosticators and patient survival. RESULTS: In our material, negative PTTG1IP immunoexpression predicted a 1.5-fold risk of breast cancer death (p = 0.02). However, adding securin immunoexpression to the analysis indicated an even stronger and independent prognostic power in the patient material (HR = 2.5, p < 0.0001). The subcellular location of securin was found with potential prognostic value also among the triple-negative breast carcinomas (n = 96, p = 0.052). CONCLUSIONS: PTTG1IP-negativity alone and in combination with high securin immunoexpression indicates a high risk of breast cancer death, resulting in up to 14-year survival difference in our material.

EGFR gene amplification is relatively common and associates with outcome in intestinal adenocarcinoma of the stomach, gastro-oesophageal junction and distal oesophagus.

BACKGROUND: Approximately 50 % of gastric adenocarcinomas belong to a molecular subgroup characterised by chromosomal instability and a strong association with the intestinal histological subtype. This subgroup typically contains alterations in the receptor tyrosine kinase-RAS pathway, for example EGFR or HER2 gene amplifications leading to protein overexpression. In clinical practice, HER2 overexpressing metastatic gastric cancer is known to respond to treatment with anti-HER2 antibodies. By contrast, anti-EGFR antibodies have not been able to provide survival benefit in clinical trials, which, however, have not included patient selection based on the histological subtype or EGFR gene copy number analysis of the tumours. To examine the role of EGFR as a potential biomarker, we studied the prevalence, clinicopathological associations as well as prognostic role of EGFR and HER2 expression and gene amplification in intestinal adenocarcinomas of the stomach, gastro-oesophageal junction and distal oesophagus. METHODS: Tissue samples from 220 patients were analysed with EGFR and HER2 immunohistochemistry. Those samples with moderate/strong staining intensity were further analysed with silver in situ hybridization to quantify gene copy numbers. The results were associated with clinical patient characteristics and survival. RESULTS: Moderate/strong EGFR protein expression was found in 72/220 (32.7 %) and EGFR gene amplification in 31/220 (14.1 %) of the tumours, while moderate/strong HER2 protein expression was detected in 31/220 (14.1 %) and HER2 gene amplification in 29/220 (13.2 %) of the tumours. EGFR and HER2 genes were co-amplified in eight tumours (3.6 %). EGFR gene amplification was more common in tumours of distal oesophagus/gastro-oesophageal junction/cardia than in those of gastric corpus (p = 0.013). It was associated with shortened time to cancer recurrence (p = 0.026) and cancer specific survival (p = 0.033). CONCLUSIONS: EGFR gene amplification is relatively common in intestinal adenocarcinomas and associates with decreased survival. It is rarely concurrent with HER2 gene amplification, suggesting that anti-EGFR therapies might be applicable to some patients not eligible for anti-HER2 treatment. Analogous to HER2 testing, determination of EGFR gene amplification status in concert with immunohistochemistry could improve the specificity of patient selection when investigating the possible benefits of anti-EGFR therapies in the treatment of gastric adenocarcinomas.

Human breast cancer cells educate macrophages toward the M2 activation status.

INTRODUCTION: The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression. METHODS: Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9). RESULTS: Clinically, we found that high numbers of CD163(+) M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type (p<0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype. CONCLUSIONS: This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome.

Association of tumor-infiltrating T lymphocytes with intestinal-type gastric cancer molecular subtypes and outcome.

While host immune response is likely to be important for the prognosis of gastric cancer patients, detailed information on the T lymphocyte infiltration in different gastric cancer subtypes is lacking. Here, we studied the presence of CD3, CD8, and FOXP3 (Forkhead box p3) expressing T lymphocytes in a retrospective cohort of 190 intestinal gastric and gastroesophageal adenocarcinomas. The cancers represented four distinct molecular subtypes: Epstein-Barr virus-positive (EBV+), mismatch-repair-deficient (MMR-D), aberrant TP53, and the "other" subtype. The absolute numbers of CD3+, CD8+, and FOXP3+ T lymphocytes were analyzed in relation with these molecular subtypes and selected clinicopathological parameters. Overall, there was a large variation in the amount of infiltrating T lymphocyte in all molecular subtypes. Among the subtypes, EBV+ cancers differed from the other subtypes in increased lymphocyte infiltration and high CD8+/FOXP3+ ratio. While the TP53 aberrant subtype did not differ in the absolute amount of T lymphocyte, the ratio of CD8+/FOXP3+ and CD3+/FOXP3+ cells was highest in this subtype, possibly reflecting immunosuppression associated with genomic instability. Increased CD3+ and CD8+ T lymphocyte infiltrates were associated with better survival, and remained as independent prognostic factors in a multivariate analysis. This study is the first to investigate lymphocytic infiltration within four molecular subtypes of intestinal-type gastric cancer in a European cohort. The results provide an important addition to the current knowledge of T lymphocyte-dependent immune response in gastric cancer and its prognostic significance.

Gastric cancer: immunohistochemical classification of molecular subtypes and their association with clinicopathological characteristics.

Gastric cancer is traditionally divided into intestinal and diffuse histological subtypes, but recent molecular analyses have led to novel classification proposals based on genomic alterations. While the intestinal- and diffuse-type tumours are distinguishable from each other at the molecular level, intestinal-type tumours have more diverse molecular profile. The technology required for comprehensive molecular analysis is expensive and not applicable for routine clinical diagnostics. In this study, we have used immunohistochemistry and in situ hybridisation in molecular classification of gastric adenocarcinomas with an emphasis on the intestinal subtype. A tissue microarray consisting of 244 gastric adenocarcinomas was constructed, and the tumours were divided into four subgroups based on the presence of Epstein-Barr virus, TP53 aberrations and microsatellite instability. The intestinal- and diffuse-type tumours were separately examined. The distribution of EGFR and HER2 gene amplifications was studied in the intestinal-type tumours. Epstein-Barr virus positive intestinal-type tumours were more common in male patients (p = 0.035) and most often found in the gastric corpus (p = 0.011). The majority of the intestinal-type tumours with TP53 aberrations were proximally located (p = 0.010). All tumours with microsatellite instability showed intestinal-type histology (p = 0.017) and were associated with increased overall survival both in the univariate (p = 0.040) and multivariate analysis (p = 0.015). In conclusion, this study shows that gastric adenocarcinomas can be classified into biologically and clinically different subgroups by using a simple method also applicable for clinical diagnostics.

EGFR gene copy number decreases during anti-EGFR antibody therapy in colorectal cancer.

Epidermal growth factor receptor (EGFR) gene copy number (GCN) increase is associated with a favorable anti-EGFR antibody treatment response in RAS wild-type metastatic colorectal cancer. However, there are limited and comparative data regarding the EGFR GCN in primary colorectal cancer tumors and corresponding metastases or the effect of anti-EGFR antibody treatment on EGFR GCN in recurrent disease. In addition, little is known about the potential EGFR GCN changes during anti-EGFR therapy in comparison with other treatment regimens. EGFR GCN was analyzed by EGFR immunohistochemistry-guided silver in situ hybridization in primary and corresponding recurrent local or metastatic tumors from 80 colorectal cancer patients. GCN levels were compared between KRAS wild-type patients having received anti-EGFR therapy and patients having received other forms of treatment after primary surgery. The EGFR GCN decrease between primary and recurrent tumors was more pronounced among the anti-EGFR-treated patients than among patients not treated with anti-EGFR therapy (P = .047). None of the patients experiencing an EGFR GCN increase of at least 1.0 between the primary and recurrent tumors were treated with anti-EGFR antibodies. When including only patients with distant metastases, an EGFR GCN decrease of at least 1.0 was more common among the anti-EGFR-treated patients than among patients not treated with anti-EGFR therapy (P = .028). Our results suggest that anti-EGFR antibody treatment is associated with EGFR GCN decrease between the primary and recurrent colorectal adenocarcinomas, whereas no GCN change is observed among patients receiving other forms of treatment after primary surgery.

Alternative splicing of human and mouse NPFF2 receptor genes: Implications to receptor expression.

Alternative splicing has an important role in the tissue-specific regulation of gene expression. Here we report that similar to the human NPFF2 receptor, the mouse NPFF2 receptor is alternatively spliced. In human the presence of three alternatively spliced receptor variants were verified, whereas two NPFF2 receptor variants were identified in mouse. The alternative splicing affected the 5' untranslated region of the mouse receptor and the variants in mouse were differently distributed. The mouse NPFF system may also have species-specific features since the NPFF2 receptor mRNA expression differs from that reported for rat.

Role of histamine H1-receptor on behavioral states and wake maintenance during deficiency of a brain activating system: A study using a knockout mouse model.

Using knockout (KO) mice lacking the histamine (HA)-synthesizing enzyme (histidine decarboxylase, HDC), we have previously shown the importance of histaminergic neurons in maintaining wakefulness (W) under behavioral challenges. Since the central actions of HA are mediated by several receptor subtypes, it remains to be determined which one(s) could be responsible for such a role. We have therefore compared the cortical-EEG, sleep and W under baseline conditions or behavioral/pharmacological stimuli in littermate wild-type (WT) and H1-receptor KO (H1-/-) mice. We found that H1-/- mice shared several characteristics with HDC KO mice, i.e. 1) a decrease in W after lights-off despite its normal baseline daily amount; 2) a decreased EEG slow wave sleep (SWS)/W power ratio; 3) inability to maintain W in response to behavioral challenges demonstrated by a decreased sleep latency when facing various stimuli. These effects were mediated by central H1-receptors. Indeed, in WT mice, injection of triprolidine, a brain-penetrating H1-receptor antagonist increased SWS, whereas ciproxifan (H3-receptor antagonist/inverse agonist) elicited W; all these injections had no effect in H1-/- mice. Finally, H1-/- mice showed markedly greater changes in EEG power (notably in the 0.8-5 Hz band) and sleep-wake cycle than in WT mice after application of a cholinergic antagonist or an indirect agonist, i.e., scopolamine or physostigmine. Hence, the role of HA in wake-promotion is largely ensured by H1-receptors. An upregulated cholinergic system may account for a quasi-normal daily amount of W in HDC or H1-receptor KO mice and likely constitutes a major compensatory mechanism when the brain is facing deficiency of an activating system. This article is part of the Special Issue entitled 'Histamine Receptors'.

The Expression of Cohesin Subunit SA2 Predicts Breast Cancer Survival.

Cohesin is one of the main regulators of sister chromatid separation during the metaphase/anaphase transition. It is a multiprotein complex consisting of 4 core subunits, one of those being the SA2 subunit. SA2 plays the final role in dismantling the cohesion complex from the sister chromatids and also functions in DNA double-strand break repair and gene regulation. There is increasing evidence regarding the involvement of both overexpression and underexpression of cohesin in cancer. Here, we present expression patterns of SA2 in different types of human breast tissue, and the prognostic analysis in the material from breast cancer patients with long-term follow-up. SA2 immunoexpression was evaluated in benign, precancerous, and malignant breast tissue, and was classified into low-intensity or high-intensity groups. The DNA content was determined by image cytometry on breast cancer cell imprints. Prognostic analyses were based on 445 breast cancer patients with upto 20 years' follow-up. SA2 immunoexpression was equally high in both benign and precancerous breast tissue. Instead, 72% of the invasive breast cancers showed deficient SA2 expression. These patients were also associated with an unfavorable outcome as indicated by a 1.6-fold risk of breast cancer death (P=0.0208). The majority (75%) of the patients with low SA2 expression were alive 6.0 years after the diagnosis, whereas the majority of the patients with high SA2 expression survived 17.6 years after the diagnosis. No statistically significant association could be detected between SA2 immunoexpression and DNA aneuploidy. Our results and previous literature indicate that decreased SA2 immunoexpression is associated with malignant breast disease and a particularly unfavorable course of disease.

Heterogeneous EGFR gene copy number increase is common in colorectal cancer and defines response to anti-EGFR therapy.

Anti-EGFR therapy is commonly used to treat colorectal cancer (CRC), although only a subset of patients benefit from the treatment. While KRAS mutation predicts non-responsiveness, positive predictive markers are not in clinical practice. We previously showed that immunohistochemistry (IHC)-guided EGFR gene copy number (GCN) analysis may identify CRC patients benefiting from anti-EGFR treatment. Here we tested the predictive value of such analysis in chemorefractory metastatic CRC, elucidated EGFR GCN heterogeneity within the tumors, and evaluated the association between EGFR GCN, KRAS status, and anti-EGFR antibody response in CRC cell lines. The chemorefractory patient cohort consisted of 54 KRAS wild-type (WT) metastatic CRC patients. EGFR GCN status was analyzed by silver in situ hybridization using a cut-off value of 4.0 EGFR gene copies/cell. KRAS-WT and KRAS mutant CRC cell lines with different EGFR GCN were used in in vitro studies. The chemorefractory CRC tumors with EGFR GCN increase (≥4.0) responded better to anti-EGFR therapy than EGFR GCN (<4.0) tumors (clinical benefit, P = 0.0004; PFS, HR = 0.23, 95% CI 0.12-0.46). EGFR GCN counted using EGFR IHC guidance was significantly higher than the value from randomly selected areas verifying intratumoral EGFR GCN heterogeneity. In CRC cell lines, EGFR GCN correlated with EGFR expression. Best anti-EGFR response was seen with KRAS-WT, EGFR GCN = 4 cells and poorest response with KRAS-WT, EGFR GCN = 2 cells. Anti-EGFR response was associated with AKT and ERK1/2 phosphorylation, which was effectively inhibited only in cells with KRAS-WT and increased EGFR GCN. In conclusion, IHC-guided EGFR GCN is a promising predictor of anti-EGFR treatment efficacy in chemorefractory CRC.

Low cdc27 and high securin expression predict short survival for breast cancer patients.

Cell cycle regulators cdc27 and securin participate in control of the mitotic checkpoint and survey the mitotic spindle to maintain chromosomal integrity. This is achieved by their functions in metaphase-anaphase transition, DNA damage repair, enhancement of mitotic arrest and apoptosis. We report on the roles of cdc27 and securin in aneuploidy and prognosis of breast cancer. The study comprises 429 breast cancer patients with up to 22 years of follow-up. DNA content was determined by image cytometry, and immunopositivity for cdc27 and securin was based on tissue microarrays. An inverse association between cdc27 and securin expression was observed in both image cytometric and immunohistochemical analyses. Low cdc27 and high securin expression identified patients with significant difference in disease outcome. Cdc27 and securin immunoexpression identified patients at risk of early cancer death within five years from diagnosis. In multivariate analysis, the combination of cdc27 and securin immunohistochemistry was the strongest predictor of cancer death after lymph node status. We demonstrate, for the first time in human breast cancer, the prognostic value of cdc27 and securin immunohistochemistry. Cdc27 and securin appear promising biomarkers for applications in predicting disease progression, prognostication of individual patients and potential in anti-mitotic drug development.

Histamine 1 receptor knock out mice show age-dependent susceptibility to status epilepticus and consequent neuronal damage.

The central histaminergic neuron system is an important regulator of activity stages such as arousal and sleep. In several epilepsy models, histamine has been shown to modulate epileptic activity and histamine 1 (H1) receptors seem to play a key role in this process. However, little is known about the H1 receptor-mediated seizure regulation during the early postnatal development, and therefore we examined differences in severity of kainic acid (KA)-induced status epilepticus (SE) and consequent neuronal damage in H1 receptor knock out (KO) and wild type (WT) mice at postnatal days 14, 21, and 60 (P14, P21, and P60). Our results show that in P14 H1 receptor KO mice, SE severity and neuronal damage were comparable to those of WT mice, whereas P21 KO mice had significantly decreased survival, more severe seizures, and enhanced neuronal damage in various brain regions, which were observed only in males. In P60 mice, SE severity did not differ between the genotypes, but in KO group, neuronal damage was significantly increased. Our results suggest that H1 receptors could contribute to regulation of seizures and neuronal damage age-dependently thus making the histaminergic system as a challenging target for novel drug design in epilepsy.

Evidence for the role of histamine H3 receptor in alcohol consumption and alcohol reward in mice.

Recent research suggests that histamine H3 receptor (H3R) antagonism may diminish motivational aspects of alcohol dependence. We studied the role of H3Rs in alcohol-related behaviors using H3R knockout (KO) mice and ligands. H3R KO mice consumed less alcohol than wild-type (WT) mice in a two-bottle free-choice test and in a 'drinking in the dark' model. H3R antagonist ciproxifan suppressed and H3R agonist immepip increased alcohol drinking in C57BL/6J mice. Impairment in reward mechanisms in H3R KO mice was confirmed by the lack of alcohol-evoked conditioned place preference. Plasma alcohol concentrations of H3R KO and WT mice were similar. There were no marked differences in brain biogenic amine levels in H3R KO mice compared with the control animals after alcohol drinking. In conclusion, the findings of this study provide evidence for the role of H3R receptor in alcohol-related behaviors, especially in alcohol drinking and alcohol reward. Thus, targeting H3Rs with a specific antagonist might be a potential means to treat alcoholism in the future.

Involvement of histamine 1 receptor in seizure susceptibility and neuroprotection in immature mice.

The central histaminergic neuronal system is a powerful modulator of brain activity, and its functional disturbance is related to e.g. epilepsy. We have recently shown in the slice culture system that histaminergic neurons attenuate kainic acid (KA)-induced epileptiform activity and neuronal damage in the hippocampus through histamine 1 (H1) receptors. We now further examined the role of H1 receptors in the regulation of KA-induced seizures and neuronal damage in immature 9-day-old H1 receptor knock out (KO) mice. In the H1 receptor KO mice, behavioral seizures were significantly more severe and duration of seizures was significantly longer when compared to the wild type (WT) mice at the KA dose of 2mg/kg. Moreover, neuronal damage correlated with seizure severity, and it was significantly increased in the thalamus and retrosplenial granular cortex (RGC) of the KO mice. The H1 receptor antagonist triprolidine treatment supported these findings by showing significantly increased seizures severity and neuronal damage in the septum, thalamus, CA3 region of the hippocampus, and RGC in the KA-treated WT mice. Our present novel findings suggest that H1 receptors play a pivotal role in the regulation of seizure intensity and duration as well as seizure-induced neuronal damage in the immature P9 mice.

Discovery of naturally occurring splice variants of the rat histamine H3 receptor that act as dominant-negative isoforms.

We described previously the cDNA cloning of three functional rat histamine H3 receptor (rH3R) isoforms as well as the differential brain expression patterns of their corresponding mRNAs and signaling properties of the resulting rH3A, rH3B, and rH3C receptor isoforms (Mol Pharmacol 59:1-8). In the current report, we describe the cDNA cloning, mRNA localization in the rat central nervous system, and pharmacological characterization of three additional rH3R splice variants (rH3D, rH3E, and rH3F) that differ from the previously published isoforms in that they result from an additional alternative-splicing event. These new H3R isoforms lack the seventh transmembrane (TM) helix and contain an alternative, putatively extracellular, C terminus (6TM-rH3 isoforms). After heterologous expression in COS-7 cells, radioligand binding or functional responses upon the application of various H3R ligands could not be detected for the 6TM-rH3 isoforms. In contrast to the rH3A receptor (rH3AR), detection of the rH3D isoform using hemagglutinin antibodies revealed that the rH3D isoform remains mainly intracellular. The expression of the rH3D-F splice variants, however, modulates the cell surface expression-levels and subsequent functional responses of the 7TM H3R isoforms. Coexpression of the rH3AR and the rH3D isoforms resulted in the intracellular retention of the rH3AR and reduced rH3AR functionality. Finally, we show that in rat brain, the H3R mRNA expression levels are modulated upon treatment with the convulsant pentylenetetrazole, suggesting that the rH3R isoforms described herein thus represent a novel physiological mechanism for controlling the activity of the histaminergic system.

Histamine H(1) and H(3) receptors in the rat thalamus and their modulation after systemic kainic acid administration.

In rat thalamus, histamine H(1) receptor and isoforms of H(3) receptor were expressed predominantly in the midline and intralaminar areas. Correspondingly, higher H(1) and H(3) receptor binding was also detected in these areas. All isoforms of H(3) receptor were expressed in several thalamic nuclei, but there were minor differences between their expression patterns. H(1) mRNA expression was high in the ventral thalamus, but the H(1) binding level was low in these areas. Since increased brain histamine appears to have an antiepileptic effect through the H(1) receptor activity, kainic acid (KA)-induced status epilepticus in rat was used to study modulation of H(1) and H(3) receptors in the thalamus following seizures. After systemic KA administration, transient decreases in mRNA expression of H(1) receptor and H(3) receptor isoforms with full-length third intracellular loops were seen in the midline areas and the H(1) receptor mRNA expression also decreased in the ventral thalamus. After 1 week, a robust increase in mRNA expression of H(3) receptor isoforms with a full-length third intracellular loop was found in the ventral posterior, posterior, and geniculate nuclei. The changes indicate a modulatory role of H(3) receptor in the sensory and motor relays, and might be involved in possible neuroprotective and compensatory mechanisms after KA administration. However, short-term increases in the H(3) receptor binding appeared earlier (72 h) than the increases of H(3) mRNA expression (1-4 w). The elevations in H(3) binding were evident in the intralaminar area, laterodorsal, lateral posterior, posterior and geniculate nuclei, and were likely to be related to the cortical and subcortical inputs to thalamus.

Transient changes in the limbic histaminergic system after systemic kainic acid-induced seizures.

Increased brain histamine is reported to protect against convulsions. We used systemic kainic acid (KA) administration to study possible changes of the histaminergic system in rat brain in status epilepticus (SE). Robust increases in brain histamine concentrations and numbers of histamine-immunoreactive nerve fibers were detected in the piriform cortex (Pir) and amygdala after KA injection, suggesting a reactive increase, which is opposite to other published aminergic transmitter responses. These changes, lasting several weeks, might be coupled to a mechanism unrelated to the anticonvulsive function of histamine. Transient increases in mRNA expression of H(3) receptor isoforms with a full-length third intracellular loop, coupled to mitogen-activated protein kinase pathway, were detected first in the hippocampal CA3c area, followed by the Pir and amygdala and then the hippocampal CA1 area. These results suggest that histamine and H3 receptors, which also control the release of GABA and glutamate, might be involved in convulsive SE.

Pain- and morphine-associated transcriptional regulation of neuropeptide FF and the G-protein-coupled NPFF2 receptor gene.

Neuropeptide FF (NPFF) is involved in pain modulation, especially plasticity during inflammatory and neuropathic pain, and opiate interactions. Its nociceptive functions may be mediated by the NPFF2 receptor. To elucidate the role of the NPFF system in plasticity associated with pathologic pain, we studied the changes of NPFF mRNA and NPFF2 receptor mRNA in rat models of acute colonic inflammation, inflammatory pain, and neuropathic pain. Furthermore, we studied the mRNA levels of both NPFF and NPFF2 receptor in morphine-tolerant rats and after acute morphine injections. We found an activation of spinal NPFF and NPFF2 receptor during early inflammatory pain. Supraspinally, we found an up-regulation of NPFF2 receptor mRNA during acute colonic inflammation and neuropathic pain. Acute, but not chronic, morphine activated the genes supraspinally. The results give further evidence for the involvement of the NPFF system in pain modulation and may provide new therapeutic opportunities for pathologic pain.