First Report of Cucumber mosaic virus Subgroup IA Isolate Infecting Yucca aloifolia in Italy.

Yucca aloifolia L. (Spanish bayonet), family Asparagaceae, is the type species of the genus Yucca. It is native to Mexico and the West Indies and is appreciated worldwide as an ornamental plant. In 2013, during a survey for viruses in ornamental plants in the Campania region of southern Italy, symptoms consisting of bright chlorotic spots and ring spots 1 to 3 mm in diameter with some necrotic streaks were observed on leaves of two plants of Y. aloifolia growing in a nursery located in the Pignataro Maggiore municipality, Caserta Province. Cucumber mosaic virus (CMV) infection was suspected because the symptoms resembled those caused by CMV in Yucca flaccida (1). A range of herbal plant indicators was inoculated with sap extracts of symptomatic Y. aloifolia plants and developed symptoms indicative of CMV. Furthermore, 30 nm isometric virus particles were observed in the same Y. aloifolia sap extracts by transmission electron microscopy. The identity of the virus was confirmed by positive reaction in ELISA tests with CMV polyclonal antisera (Bioreba) conducted on sap extracts of symptomatic Y. aloifolia plants and systemically infected symptomatic hosts (i.e., Nicotiana tabacum, N. glutinosa, Cucumber sativus cv. Marketer, Solanum lycopersicum cv. San Marzano). The presence of CMV in the two naturally infected Y. aloifolia and other mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with the E.Z.N.A. Plant RNA Kit (Omega Bio-Tek), according to the manufacturer's instructions. RT-PCR was carried out with the ImProm-II Reverse Transcription System first-strand synthesis reaction (Promega) using the primer pair CMV1 and CMV2 (2). These primers amplify part of the CP gene and part of the 3'-noncoding region of CMV RNA3 and were designed to produce amplicons of different sizes to distinguish CMV isolates belonging to subgroups I or II (3). RT-PCR products were obtained from both naturally infected Y. aloifolia and mechanically inoculated plants as well as from PAE1 isolate of CMV (2), used as positive control, but not from healthy plants. Based on the length of the amplicons obtained (487 bp), the CMV isolate from Y. aloifolia (named YAL) belonged to subgroup I (3). The amplified RT-PCR products were purified with QIAquick PCR Purification Kit (Qiagen), cloned in the pGEMT vector (Promega), and three independent clones were sequenced at MWG (Ebersberg, Germany). Sequences obtained from the two CMV-infected Y. aloifolia plants were identical. This sequence was deposited at GenBank (Accession No. HG965199). Multiple alignments of the YAL sequence with sequences of other CMV isolates using MEGA5 software revealed highest percentage of identity (98.9%) with the isolates Z (AB369269) and SO (AF103992) from Korea and Japan, respectively. Moreover, the YAL isolate was identified as belonging to subgroup IA, based on the presence of only one HpaII restriction site in the 487-bp sequence, as previously proposed (2). Although CMV seems to not be a major threat currently for the production of Y. aloifolia, because the farming of this plant is performed using vegetative propagation, particular attention should be given to the presence of the virus in donor mother plants in order to avoid the dispersion of infected plants that could serve as sources for aphid transmission to other susceptible plant species. To our knowledge, this is the first report of CMV infection of Y. aloifolia in the world. References: (1) I. Bouwen et al. Neth. J. Plant Pathol. 84:175, 1978. (2) G. Parrella and D. Sorrentino. J. Phytopathol. 157:762, 2009. (3) Z. Singh et al. Plant Dis. 79:713, 1995.

Dieback and Wilting Caused by Tomato spotted wilt virus in Arctotis × hybrida in Italy.

In winter 2012, some potted plants of African daisy (Arctotis × hybrida L., family Asteraceae) cv. Hannah, propagated by rooted stem cuttings and cultivated for commercial purposes in a greenhouse located at Albenga (Liguria region, Italy), were noticed for a rapid dieback, generalized reddening, following by an irreversible wilting. Around 130 plants on a total of 3,000 cultivated plants showed symptoms (4 to 5%). One gram of fresh leaves, each collected from three different symptomatic plants, was ground in 4 ml of cold (∼5°C) sodium phosphate 0.03 M buffer, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of carborundum (600 mesh). The inoculum was rubbed on healthy indicator herbaceous plants and inoculated plants were maintained in an insect-proof greenhouse with natural illumination and temperatures of 24/18°C day/night. Healthy and buffer inoculated plants were also included in the test and used as negative control in the subsequent serological and molecular analysis. Sap-inoculated plants showed the following symptoms after 1 to 3 weeks: necrotic local lesions in Chenopodium amaranticolor and C. quinoa, yellowing and stunting following by systemic necrosis and death of the plants in tomato (Solanum lycopersicum cv. San Marzano), necrotic local lesions following by systemic necrotic patterns and leaf deformation in tobacco (Nicotiana tabacum cv. Xanthi nc.) and N. glutinosa, necrotic local lesions in petunia (Petunia × hybrida cv. Pink Beauty). No symptoms were recorded on buffer inoculated plants. Leaf samples from both symptomatic hosts and the three original symptomatic African daisy plants were tested by double-antibody sandwich-ELISA with polyclonal antisera against Cucumber mosaic virus (CMV) and tospoviruses (Tospovirus broad-spectrum, Serogroups I, II, and III, Bioreba AG, Switzerland). Positive reaction was obtained with Tospo-groups antibodies, but not with the CMV ones. Total RNA was extracted from infected leaves of African daisy with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to reverse transcription (RT)-PCR by using the tospovirus universal primers BR60/BR65 that amplify part of the nucleocapsid protein gene (1). Target amplicons of 454 bp were produced for all samples tested. The PCR products were cloned and sequenced on both strands (one clone per amplicon cloned). The resulting sequences were 100% identical, so a single sequence was deposited in GenBank (HF913777). The sequence showed highest homology (99%) with the Tomato spotted wilt virus (TSWV) tomato isolate NJ-JN from South Korea (HM581936). The identity of the virus infecting African daisy was further confirmed by sequencing amplicons obtained by RT-PCR using primers partially covering the movement protein gene of TSWV (2). The sequence obtained (HF913776) showed the highest homology (99%) with three TSWV isolates: a tomato isolate from Spain (AY744493), a pepper isolate from South Korea (AB663306), and again the tomato NJ-JN isolate from South Korea (HM581936). To our knowledge, this is the first natural report of TSWV infecting African daisy plants. Moreover, since this ornamental is often cultivated with other flowering plants, it can act as reservoir for the virus that can infect other ornamentals and crops, considering that TSWV has a very broad host range (3). This result also represents the first finding of TSWV in the genus Arctotis, family Asteraceae, the greater botanical family of TSWV hosts (3). References: (1) M. Eiras et al. Fitopatol. Bras. 26:170, 2001. (2) M. M. Finetti et al. J. Plant Pathol. 84:145, 2002. (3) G. Parrella et al. J. Plant Pathol. 85:227. 2003.

Araujia sericifera New Host of Alfalfa mosaic virus in Italy.

Araujia sericifera Brot. (Fam. Apocynaceae) is an evergreen climbing plant native of South America, originally introduced in Europe as an ornamental. In spring 2012, virus-like symptoms including bright yellow mosaic of calico-type and leaf distortion were observed in three A. sericifera plants growing in an abandoned field located in Pomigliano d'Arco (Campania region, Italy). Leaves from the three plants were collected and examined using commercial antisera (Bioreba AG, Reinach, Switzerland) by double antibody sandwich (DAS)-ELISA against Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV), and by indirect plate trapped antigen (PTA)-ELISA against potyviruses (Potygroup test). Only AMV was detected serologically in the three A. sericifera samples. The virus was mechanically transmitted from the ELISA-positive samples to four plants each of Chenopodium quinoa, C. amaranticolor, tobacco (Nicotiana tabacum cv. Xanthi nc), cowpea (Vigna unguiculata, cv. Black eyes), basil (Ocimum basilicum, cv. Gigante), and tomato (Solanum lycopersicum cv. San Marzano), using chilled 0.03 M sodium phosphate buffer, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of Carborundum (600 mesh). Inoculated plants were kept in an insect-proof greenhouse with natural illumination and temperatures of 24 and 18°C day/night. Under these conditions, plants showed the following symptoms after 1 to 3 weeks, consistent with symptoms caused by AMV (1): chlorotic local lesions following by mosaic in C. quinoa and C. amaranticolor, reddish local lesions following by mosaic in cowpea, necrotic local lesions followed by systemic necrosis in tomato, bright yellow mosaic (calico type) in basil, and mosaic and strong deformation of the apical leaves in tobacco. The presence of AMV in ELISA-positive A. sericifera and host plants was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using primers for the coat protein gene (CP) previously used for the molecular characterization of AMV isolates (2). An Italian isolate of AMV from Lavandula stoechas (GenBank Accession No. FN667967) and RNA extracted from a healthy A. sericifera plant were used as positive and negative controls, respectively. An amplicon of the correct predicted size (∼750 bp) was obtained from each of the infected plants assayed, and that derived from A. sericifera isolate Ars2 was purified (QIAqick PCR Purification Kit, Qiagen), cloned in pGEMT easy vector (Promega, Fitchburg, WI) and sequenced (HF570950). Sequence analysis of the CP gene, conducted with MEGA5 software, revealed the highest nucleotide identity of 98% (99% amino acid identity) with the AMV isolate Tef-1 (FR854391), an isolate belonging to subgroup I (3). To our knowledge, this is the first report of AMV infecting A. sericifera in Italy. Since A. sericifera is considered an invasive plant, in continuous expansion to new areas in Italy and in other European countries, particular attention should be paid to the possibility that this species may play a role in the epidemiology of aphid-transmitted viruses such as AMV and CMV, representing a threat to susceptible crops growing nearby. References: (1) G. Marchoux et al. Page 163 in: Virus des Solanacées. Quae éditions, Versailles, 2008. (2) G. Parrella et al. Arch. Virol. 145:2659, 2000. (3) G. Parrella et al. Plant Dis. 96:249, 2012.

Identificación de especies cárnicas en productos cárnicos cocidos: utilización de sds-page como método de screening / Meat species identification in cooked meat products: using sds-page as screening method

It is necessary to have methodologies for the detection of meat species used in meat products in order to establish possible adulterations. Twenty cooked meat products produced in pilot plants or commercially available products were analyzed in order to evaluate the SDS-PAGE electrophoresis methodology as a screening method to identify the meat species used. The results found by electrophoresis were compared with an immunochemical method (ELISA kits for detection ofpork, beef and poultry). SDS-PAGE methodology allowed the detection of beef, pork, chicken and /or turkey proteins in most samples. When some of these species were present in low proportion this methodology was not able to detect them. SDS-PAGE method has the advantage that allows the detection of proteins of different meat species in only one electrophoresis run while with the ELISA method it is necessary to analyze the same sample with different species identification kits to confirm the presence of the species used.

Es necesario contar con metodologías que permitan la detección de las especies cárnicas utilizadas en la elaboración de productos cárnicos a los fines de establecer posibles adulteraciones. Con la finalidad de evaluar SDS-PAGE como método de screening para identificar la/las especies cárnicas utilizadas" en el presente trabajo" se analizaron veinte productos cárnicos crudos o cocidos elaborados en plantas piloto o comerciales. Los resultados hallados por electroforesis se compararon con un método inmunoquímico (ELISA). SDS-PAGE permitió la detección de proteínas de carne vacuna" porcina" de pollo y/o de pavo en las mayoría de las muestras analizadas. Sólo en algunas muestras que contenían alguna de las especies cárnicas en baja proporción" esta metodología no permitió su detección. SDS-PAGE tiene como ventaja poder detectar en una sola corrida la presencia de proteínas de diferentes especies cárnicas" mientras que con ELISA es necesario analizar una misma muestra con los diferentes kits de especies cárnicas para confirmar la presencia de las especies utilizadas.

Macrophages and liposomes in inflammatory disease: friends or foes?

Liposome-encapsulated corticosteroids have shown to exert strong beneficial effects in inflammatory diseases, such as arthritis and cancer. To extend the clinical applicability of these potent nanomedicines, the therapeutic effect of dexamethasone phosphate loaded long-circulating liposomes (LCL-DXP) was evaluated in animal models of multiple sclerosis (MS) and Crohn's disease (CD). In mice with experimental autoimmune encephalitis (EAE), a model for MS, treatment with LCL-DXP, but not free DXP, resulted in a decrease in disease activity when compared to PBS treated mice. In contrast, in mice with chronic DSS-induced colitis, a model for CD, treatment with LCL-DXP did not induce an improvement, but in fact worsened the fecal blood loss after treatment, indicating an aggravation of the disease. It is hypothesized that modulation of macrophage polarization towards a M2 phenotype underlies the efficacy of corticosteroid-based drug delivery systems, which is supported by the presented data. On the one hand, M1 polarized macrophages are part of the pathogenesis of MS; the modulation to M2-polarization by LCL-DXP is therefore beneficial. On the other hand, M1-polarized intestinal macrophages fulfill a protective and inflammation-suppressing role in intestinal homeostasis; changing their phenotype to M2 causes reduced protection to invading microorganisms, leading to a more severe intestinal inflammation. These findings therefore indicate that the interplay between the specific phenotype of macrophages and the specific inflammatory context of the inflammatory disease in question may be an important determining factor in the therapeutic applicability of liposomal corticosteroids in inflammatory disease.

Combined Percutaneous and Endovascular Treatment of Symptomatic Aneurysmal Bone Cyst of the Spine: Clinical Six Months. Follow-up of Six Cases.

We describe the usefulness of endovascular and direct percutaneous treatment as a therapy option for aneurysmal bone cysts (ABCs) of the spine. From January 2007 to December 2008, we treated six consecutive patients with symptomatic ABCs resistant to continuous medical management or with acute clinical onset of paraparesis at cervical, thoracic and lumbar spine level. Two patients were treated after emergency laminectomy. All patients were studied with an MRI protocol and multidetector CT with MPR reconstructions followed by angiographic control before treatment. The procedure was performed under general anaesthesia for all patients. Under CT or fluoroscopy guidance, percutaneous treatment was performed either by direct injection of Glubran(®) diluted at 30% with Lipiodol(®) only, or combined with endovascular treatment by Onyx® injection. Clinical and X-ray follow-up was performed at three and six months. Combined endovascular and percutaneous treatment for ABCs was successful and led to an excellent outcome in five out of six patients with clinical improvement. There were no periprocedural or subsequent clinical complications and the glue resulted in successful selective permanent occlusion with intralesional penetration. Direct sclerotherapy resulted in immediate thrombosis of the malformation with no progression of symptoms. Complete healing was observed in five out of six aggressive lesions. No major complications were noted. At six month follow-up the symptoms had completely resolved and X-ray control showed a partial or total sclerotic reaction of the lesion with stable clinical results (no partial or clinical abnormalities). One patient had a recurrence of the ABC with spinal cord cervical clinical symptomatology. Combined endovascular and percutaneous treatment or direct percutaneous sclerotherapy with glue alone are important, safe, effective therapy options for symptomatic aneurysmal bone cyst. Results are stable and confirmed by clinical and X-ray follow-up six months after treatment.

Administration of a monomeric CCL2 variant to EAE mice inhibits inflammatory cell recruitment and protects from demyelination and axonal loss.

Based on gene expression data, we tested the P8A-CCL2 variant of the chemokine CCL2, able to interfere with the chemotactic properties of the parental molecule, in relapsing-remitting (RR)-EAE SJL. Only preventive treatment significantly delayed disease onset in a dose dependent manner. P8A-CCL2 administration, however, decreased demyelination, axonal loss and number of CNS infiltrating T cells and macrophages. Immunological analysis revealed that P8A-CCL2 does not act on Ag-specific T cell proliferation and does not interfere with the differentiation of IFNgamma-releasing effectors T cells. These results suggest that the therapeutic mechanism of P8A-CCL2 may rely on interference with immune cell recruitment.

Beneficial effects of r-h-CLU on disease severity in different animal models of peripheral neuropathies.

Clusterin is a protein involved in multiple biological events, including neuronal cytoprotection, membrane recycling and regulation of complement-mediated membrane attack after injury. We investigated the effect of recombinant human clusterin in preclinical models of peripheral neuropathies. Daily treatment with clusterin accelerated the recovery of nerve motor evoked potential parameters after sciatic nerve injury. Prophylactic or therapeutic treatment of experimental autoimmune neuritis rats with clusterin also accelerated the rate of recovery from the disease, associated with remyelination of demyelinated nerve fibers. These data demonstrate that clusterin is capable of ameliorating clinical, neurophysiological and pathological signs in models of peripheral neuropathies.

Complete nodal staging in male breast cancer. (Biopsy of axillary and internal mammary sentinel node).

Carcinoma of the male breast is an uncommon phenomenon, accounting for < 1% of all malignancies in men. Searching for a more conservative treatment we introduced in our clinical practice axillary sentinel node biopsy and, if present, sentinel node biopsy of the internal mammary chain. The potential clinical implications of complete nodal staging are far-reaching, and give us a major new opportunity to stratify male patients with breast cancer for appropriate surgery as well as giving valuable prognostic information.

Response to male odours in progestin receptor- and oestrogen receptor-containing cells in female rat brain.

Sensory cues from male rats, such as odours and vaginal-cervical stimulation (VCS), play a modulatory role in female rat sexual behaviour. For example, exposure to male odours and VCS appears to be at least partially responsible for increases in sexual behaviour following repeated mating of oestradiol-primed female rats. Although there is evidence that VCS influences sexual behaviour via a ligand-independent progestin receptor (PR)-dependent mechanism, the mechanism by which odours influence sexual behaviour is not known. We tested the hypothesis that, similar to VCS, the effects of male odours on sexual behaviour are mediated by progestin receptors. Female rats were injected with the progestin antagonist, RU486, or oil vehicle and were then exposed to male-soiled bedding or clean bedding. Although exposure to male-soiled bedding resulted in higher levels of Fos immunoreactivity in brain areas associated with female sexual behaviour, the progestin antagonist did not reduce this effect. Furthermore, there was minimal coexpression of odour-induced Fos and progestin receptors in brain areas associated with female sexual behaviour. Together, these results suggest that the effects of male odours are not mediated by a PR-dependent mechanism. Therefore, we tested the hypothesis that oestrogen receptor (ER)-containing cells are involved in the effects of olfactory cues. Although there was virtually no coexpression of ERbeta and odour-induced Fos in brain areas associated with female sexual behaviour, exposure to male odours slightly increased the number of cells coexpressing ER(alpha) and odour-induced Fos in the posterodorsal medial amygdala. Although, these results do not support the hypothesis that the effects of odours are mediated by a PR-dependent mechanism, they suggest that integration of male odours and hormonal cues may occur in ER(alpha)-containing cells in the posterodorsal medial amygdala.

A progestin antagonist blocks vaginocervical stimulation-induced fos expression in neurones containing progestin receptors in the rostral medial preoptic area.

Vaginocervical stimulation (VCS) has a variety of effects on the brain, physiology and behaviour. Previous work demonstrated that a progestin antagonist blocked neuronal response to VCS (i.e. Fos expression) in the absence of progesterone in some neurones, and suggested that some of the effects of VCS on the brain are mediated by ligand-independent activation of progestin receptors (PRs). Although it had been reported previously that some of the cells in which VCS induces Fos expression also contain PRs, it had not been determined if a progestin antagonist blocked Fos expression in these particular neurones. The purpose of this experiment was to determine if a progestin antagonist decreases Fos expression specifically in cells that also express PRs in the preoptic area and ventromedial hypothalamus. As has been shown previously, VCS increased Fos-immunoreactive (ir) expression in the particular areas studied. In the rostral medial preoptic area, VCS increased Fos expression in cells that coexpressed PRs, as well as in cells that do not. However, in the caudal medial preoptic area, VCS only increased Fos expression in cells that did not coexpress PRs. Injection of the progestin antagonist, RU 486, decreased Fos expression in the rostral, but not caudal medial preoptic area, and it decreased Fos expression only in cells that coexpressed PR-ir. In contrast to a previous report, in the present study, the progestin antagonist did not inhibit VCS-induced Fos expression in the ventromedial hypothalamic area. The results of this experiment suggest that the progestin antagonist inhibits VCS-induced Fos expression in some neurones by blocking PRs, and they provide further support for the idea that VCS influences neuronal response in some cells by ligand-independent activation of PRs in those cells.

Coexpression of ER beta with ER alpha and progestin receptor proteins in the female rat forebrain: effects of estradiol treatment.

Estrogen and progestin receptors (ER, PgR) play a critical role in the regulation of neuroendocrine functions in females. The neuroanatomical distribution of the recently cloned, ER beta, overlaps with both ER alpha and PgR. To determine whether ER beta is found within ER alpha- or PgR-containing neurons in female rat, we used dual label immunocytochemistry. ER beta-immunoreactivity (ER beta-ir) was primarily detected in the nuclei of cells in the periventricular preoptic area (PvPO), the bed nucleus of the stria terminalis (BNSTpr), the paraventricular nucleus, the supraoptic nucleus, and the medial amygdala (MEApd). Coexpression of ER beta-ir with ER alpha-ir or PgR-ir was observed in the PvPO, BNSTpr, and MEApd in ovariectomized rats. E2 treatment decreased the number of ER beta-ir cells in the PvPO and BNSTpr and the number of ER alpha-ir cells in the MEApd and paraventricular nucleus, and therefore decreased the number of cells coexpressing ER beta-ir and ER alpha-ir in the PvPO, BNSTpr, and MEApd. E2 treatment increased the amount of PgR-ir in cells of the PvPO, BNSTpr, and MEApd, a portion of which also contained ER beta. These results demonstrate that ER beta is expressed in ER alpha- or PgR-containing cells, and they suggest that E can modulate the ratios of these steroid receptors in a brain region-specific manner.

Neuropeptide Y inhibits estrous behavior and stimulates feeding via separate receptors in Syrian hamsters.

Central injections of neuropeptide Y (NPY) increase food intake in Syrian hamsters; however, the effect of NPY on sexual behavior in hamsters is not known nor are the receptor subtypes involved in feeding and sexual behaviors. We demonstrate that NPY inhibits lordosis duration in a dose-related fashion after lateral ventricular injection in ovariectomized, steroid-primed Syrian hamsters. Under the same conditions, we compared the effect of two receptor-differentiating agonists derived from peptide YY (PYY), PYY-(3-36) and [Leu(31),Pro(34)]PYY, on lordosis duration and food intake. PYY-(3-36) produced a 91% reduction in lordosis duration at 0.24 nmol. [Leu(31),Pro(34)]PYY was less potent, producing a reduction in lordosis duration (66%) only at 2.4 nmol. These results suggest NPY effects on estrous behavior are principally mediated by Y2 receptors. PYY-(3-36) and [Leu(31),Pro(34)]PYY stimulated comparable dose-related increases in total food intake (2 h), suggesting Y5 receptors are involved in feeding. The significance of different NPY receptor subtypes controlling estrous and feeding behavior is highlighted by results on expression of Fos immunoreactivity (Fos-IR) elicited by either PYY-(3-36) or [Leu(31),Pro(34)]PYY at a dose of each that differentiated between the two behaviors. Some differences were seen in the distribution of Fos-IR produced by the two peptides. Overall, however, the patterns of expression were similar. Our behavioral and anatomic results suggest that NPY-containing pathways controlling estrous and feeding behavior innervate similar nuclei, with the divergence in pathways controlling the separate behaviors characterized by linkage to different NPY receptor subtypes.

Maternal behavior stimulates c-fos activity within estrogen receptor alpha-containing neurons in lactating rats.

Estradiol and other hormones are thought to be critical for the onset, but not maintenance, of maternal behavior in rats. Maternal behavior is instead maintained postpartum by tactile stimulation that dams receive during interactions with pups, and many neural sites implicated in the control of maternal behavior show elevated c-fos activity in response to this stimulation. Many of these sites also contain neurons that express the alpha subtype of the estrogen receptor (ERalpha). Because of possible interactions between tactile stimulation from pups, c-fos, and ERalpha in the lactating rat brain, we determined if populations of cells that show increased c-fos activity after maternal behavior in lactating rats also contain ERalpha. Dams were separated from their pups for 48 h beginning on day 5 postpartum. On day 7 postpartum, experimental dams were reunited with pups and mother-litter interactions were observed for 60 min. Control dams received no pup stimulation. Subjects were sacrificed 60 min later and brain sections were double immunolabeled for the Fos and ERalpha proteins. As expected, the number of ERalpha-immunoreactive (ERalpha-ir) neurons did not differ between the two groups in the eight areas analyzed (lateral region of the lateral septum, posterodorsal medial amygdala, dorsal and ventral medial preoptic area, dorsal and ventral bed nucleus of the stria terminalis, lateral habenula, and ventrolateral caudal periaqueductal gray). Consistent with previous reports, maternal dams had 2- to 7-fold more Fos-immunoreactive (Fos-ir) neurons in these sites compared with nonstimulated controls. Maternal dams had significantly more Fos-ir neurons that also contained ERalpha-ir in all sites, with the greatest increases in the ventral medial preoptic area, lateral habenula, and ventral bed nucleus of the stria terminalis. Between approximately 25 and 45% of the Fos-ir cells in the sites examined also expressed ERalpha. Thus, a substantial number of neurons that are genomically activated during maternal behavior contain ERalpha, raising the possibility that the postpartum display of maternal behavior can be influenced by ERalpha activity.

Colocalization of androgen receptors and mating-induced FOS immunoreactivity in neurons that project to the central tegmental field in male rats.

Bilateral lesions of the central tegmental field (CTF) in male rats virtually eliminate mating behavior. This study examined if mating-induced Fos expression (a measure of neuronal activation) and androgen receptors (AR) are colocalized in brain and spinal cord neurons which project to the CTF. Animals received unilateral injections of the retrograde tracer Fluorogold (FG) in the lateral part of the CTF (CTFl), and 10 days later were killed after ejaculating with females. Brains and spinal cords were examined for FG transport, AR-immunoreactivity (AR-ir), and Fos-immunoreactivity (Fos-ir). AR-ir and Fos-ir were visualized with fluorescence microscopy using cyanine-conjugated and fluorescein-conjugated secondary antibodies. The CTFl received projections from AR-containing neurons in forebrain structures (bed nucleus of stria terminalis, medial preoptic area, lateral and ventromedial hypothalamus), in the central amygdala and various mid- and hindbrain structures (dorsolateral tegmentum, superior and inferior colliculi, pedunculopontine nucleus), and in the lumbosacral spinal cord (lamina X). Some of the AR-containing neurons in bed nucleus of stria terminalis and in the dorsal part of the medial preoptic area with projections to the CTFl were activated by mating. Most AR-containing neurons in spinal lamina X with projections to the CTFl were also activated by mating. Information from spinal cord and pontine nuclei and from outputs descending from the forebrain may be relayed in the CTFl. Thus, as part of a network of hormone-sensitive neurons linking brain and spinal cord mechanisms for mating, the CTFl could participate in the integration of visceral and somatic information relevant for sexual behavior.

Impairment of phagocytic and T-cell-mediated antibacterial activity and plasma endotoxins in patients with untreated gastrointestinal cancer.

BACKGROUND: Cancer patients have multiple immune deficits, and mediators, such as prostaglandins, transforming growth factor-beta, and interleukin (IL)-10, may play a role in the pathogenesis of these immune dysfunctions. METHODS: Fifty-six patients with gastrointestinal cancer (11 gastric cancer, 7 papilla of Vater cancer, and 38 colorectal cancer) were enrolled for this study, before starting conventional treatments. Phagocytosis and killing exerted by polymorphonuclear cells and monocytes, peripheral blood mononuclear cell absolute numbers, T-cell-mediated antibacterial activity, serum levels of IL-10 and interferon (IFN)-gamma, and plasma bacterial endotoxin concentration were evaluated. RESULTS: Data show an impaired phagocytic and T-cell-mediated antibacterial activity in all cancer patients, whereas only in subjects with gastric cancer were IFN-gamma serum levels reduced. Circulating endotoxins were detected in 17 patients. CONCLUSIONS: In untreated gastrointestinal cancer patients the capacity of phagocytes and T-cells to clear pathogens is reduced. This dysfunction may increase the risk of becoming infected and may account for the presence of endotoxin in 30% of patients.

Fos induced by mating or noncontact sociosexual interaction is colocalized with androgen receptors in neurons within the forebrain, midbrain, and lumbosacral spinal cord of male rats.

This study was designed to determine the extent to which Fos immunoreactivity (induced either by mating or noncontact sociosexual interaction) and androgen receptor (AR) immunoreactivity are colocalized in brain and spinal cord of male rats. Some males (Mated) were allowed to mate to ejaculation; others (Social Controls) were placed with females but physical contact was prevented by a wire mesh screen; remaining males (Isolated) were placed alone in the test jar for the duration of the test period. After testing, brains and spinal cords were examined for AR and Fos immunoreactivity (ir). PG21 anti-AR and anti-c-fos primary antibodies were visualized by fluorescence microscopy using cyanine-conjugated and fluorescein-conjugated secondary antibodies. In both brain and spinal cord, the number of Fos-ir neurons varied according to group: Mated males > Social Controls > Isolated males. Fos was highly localized in subsets of AR-ir neurons within the medial preoptic nucleus, bed nucleus of the stria terminalis, dorsomedial nucleus of the amygdala, and central tegmental field. Fos was also localized in subsets of AR-ir neurons within the L5, L6, and S1 segments of the spinal cord. Spinal cord concentrations of AR-ir and Fos-ir neurons were greatest in Lamina X, and the vast majority of Fos-ir neurons in the dorsal part of Lamina X were also AR-ir. Thus, in both brain and spinal cord, androgen-sensitive neurons are active during mating, and transmission of sexually relevant information from cord to brain is probably accomplished via hormone-sensitive spinal neurons.

Androgen receptor and mating-induced fos immunoreactivity are co-localized in limbic and midbrain neurons that project to the male rat medial preoptic area.

Two studies were designed to document neuronal colocalization of androgen receptor immunoreactivity and mating-induced Fos immunoreactivity (AR-ir, Fos-ir) in brain of male rats and to examine the extent to which limbic and midbrain neurons that project to the preoptic area are androgen sensitive and activated by mating. Brains from male rats, killed 1 h after ejaculating with receptive females, were examined for Fos-ir and AR-ir and compared with those from control rats not given access to females. PG21 anti-AR and anti-c-fos primary antibodies were visualized by fluorescence microscopy using cyanine-conjugated and fluorescein-conjugated secondary antibodies. In mated males (Expt. 1), Fos-ir and AR-ir were colocalized in neurons of the medial preoptic nucleus (MPN), the dorsal medial amygdala (dMEA), the central tegmental field (CTF), the bed nucleus of the stria terminalis, the anterior hypothalamus, the lateral hypothalamus, and the ventral premamillary nucleus. In Expt. 2, male rats received a unilateral injection of the retrograde tracer FluoroGold (FG) in the preoptic area and four days later were killed after ejaculating with receptive females. Brains were subsequently examined for FG transport, Fos-ir and AR-ir. Fluorogold-containing neurons were present in dMEA and CTF as well as in other hypothalamic and limbic regions known to project to the MPN. In dMEA and CTF, nuclear colocalization of AR-ir and mating-induced Fos-ir was present in a proportion of FG-containing neurons. Sexually relevant information may be carried through the brain by an interconnected network of hormone-sensitive neurons.

Androgen receptors and estrogen receptors are colocalized in male rat hypothalamic and limbic neurons that express Fos immunoreactivity induced by mating.

Conversion of testosterone into estradiol is important for male rat sexual behavior, and both steroids probably contribute to mating. The distributions of neurons containing androgen receptors (AR) and estrogen receptors (ER) overlap, and many AR-immunoreactive (AR-ir) neurons express Fos immunoreactivity (Fos-ir) induced by mating. Because mating-induced Fos-ir in the male rat occurs mainly in AR-ir neurons, and because both steroids are important for mating, we hypothesized that (i) AR-ir and ER-ir are colocalized and that (ii) some of these neurons are activated during mating. We examined, in adjacent sections from the medial preoptic area (MPN) through the central tegmental field (CTF), the expression of ER-ir in: (i) AR-ir-containing neurons, and (ii) Fos-ir-expressive neurons. PG21 anti-AR, OA-11-824 anti-c-fos, H222 or 1D5 anti-ER primary antibodies were visualized, respectively, with cyanine-conjugated, fluorescein- or cyanine-conjugated, and fluorescein-conjugated secondary antibodies in male rats which were killed 1 h after ejaculating with a receptive female. In MPN, bed nucleus of the stria terminalis (BNST), and medial amygdala (MEA), 80-90% of ER-ir labeling occurred in AR-ir-positive neurons but only about 30% of AR-ir neurons were ER-ir-positive. No ER-ir was found in the CTF. This suggests the presence of three types of brain neurons sensitive to gonadal steroid hormones: neurons sensitive to androgens only, neurons sensitive to both androgens and estrogens, and neurons sensitive to estrogens only. About 50% of ER-ir labeling occurred in cells expressing mating-induced Fos-ir but only about 30% of Fos-ir neurons were ER-ir-positive. These findings suggest that, in the MPN, at least two different neuronal populations are activated during mating: the first contains AR-ir only and the second contains AR-ir and ER-ir. In the BNST and MEA, at least three hormonally sensitive populations are activated during mating: the two described above plus a third population which expresses ER-ir only.