Localization of metastasis within the sentinel lymph node biopsies: a predictor of additional axillary spread of breast cancer?

PURPOSE: To explore the relationship between morphological characteristics and histologic localization of metastasis within sentinel lymph nodes (SLN) and axillary spread in women with breast cancer. METHODS: We selected 119 patients with positive SLN submitted to complete axillary lymph node dissection from July 2002 to March 2007. We retrieved the age of patients and the primary tumor size. In the primary tumor, we evaluated histologic and nuclear grade, and peritumoral vascular invasion (PVI). In SLNs we evaluated the size of metastasis, their localization in the lymph node, number of foci, number of involved lymph nodes, and extranodal extension. RESULTS: Fifty-one (42.8%) patients had confirmed additional metastasis in non-sentinel lymph nodes (NLSN). High histologic grade, PVI, intraparenchymatous metastasis, extranodal neoplastic extension and size of metastasis were associated with positive NLSN. SLN metastasis affecting the capsule were associated to low risk incidence of additional metastasis. After multivariate analysis, PVI and metastasis size in the SLN remained as the most important risk factors for additional metastasis. CONCLUSIONS:The risk of additional involvement of NSLN is higher in patients with PVI and it increases progressively according the histologic localization in the lymph node, from capsule, where the afferent lymphatic channel arrives, to the opposite side of capsule promoting the extranodal extension. Size of metastasis greater than 6.0 mm presents higher risk of additional lymph node metastasis.

Field evaluation of safety during gestation and horizontal spread of a recombinant differential bovine herpesvirus 1 (BoHV-1) vaccine

Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of respiratory, reproductive disease and abortion in cattle. Vaccination is widely applied to minimize losses induced by BoHV-1 infections; however, vaccination of dams during pregnancy with modified live virus (MLV) vaccines has been occasionally associated to abortions. We have previously reported the development of a BoHV-1 recombinant virus, constructed with basis on a Brazilian BoHV-1 (Franco et al. 2002a) from which the gene coding for glycoprotein E (gE) was deleted (gE-) by genetic manipulation. Such recombinant has been previously evaluated in its potential as a differential vaccine (gE- vaccine) that allows differentiation between vaccinated and infected animals. Here, in the first part of the present study, the safety of the gE- vaccine during pregnancy was evaluated by the intramuscular inoculation of 10(7.4) tissue culture 50 % infective doses (TCID50) of the virus into 22 pregnant dams (14 BoHV-1 seronegative; 8 seropositive), at different stages of gestation. Other 15 pregnant dams were kept as non-vaccinated controls. No abortions, stillbirths or fetal abnormalities were seen after vaccination. Seroconversion was observed in both groups of previously seronegative vaccinated animals. In the second part of the study, the potential of the gE- vaccine virus to spread among beef cattle under field conditions was examined. Four heifers were inoculated intranasally with a larger amount (10(7,6) TCID50) of the gE- vaccine (to increase chances of transmission) and mixed with other sixteen animals at the same age and body condition, in the same grazing area, at a population density equal to the average cattle farming density within the region (one cattle head per 10,000 m²), for 180 days. All animals were monitored daily for clinical signs. Serum samples were collected on days 0, 30, 60 and 180 post-vaccination. Seroconversion was observed only in vaccinated heifers. These results indicate that, under the conditions of the present study, the gE- vaccine virus did not cause any noticeable harmful effect on pregnant dams and on its offspring and did not spread horizontally among cattle.

Molecular epidemiology and extended-spectrum β-lactamases production of Klebsiella pneumoniae isolated from three dairy herds

The objectives of this study were to isolate Klebsiella pneumoniae from different sources in three dairy cattle herds, to use the pulsed-field gel electrophoresis (PFGE) to measure genotypic similarities between isolates within a dairy herd, to verify the production of extended-spectrum β-lactamases (ESBLs) by the double-disk synergy test (DDST), and to use the PCR to detect the main ESBLs subgroups genes. Three dairy farms were selected based on previous mastitis outbreaks caused by K. pneumoniae. Milk samples were collected from lactating cows and from the bulk tank. Swabs were performed in different locations, including milking parlors, waiting room, soil, animal's hind limbs and rectum. K. pneumoniae was isolated from 27 cases of intramammary infections (IMI) and from 41 swabs. For farm A isolates from IMI and bulk tank were considered of the same PGFE subtype. One isolate from a bulk tank, three from IMI cases and four from environmental samples were positive in the DDST test. All eight DDST positive isolates harbored the bla shv gene, one harbored the bla tem gene, and three harbored the bla ctx-m gene, including the bulk tank isolate. Our study confirms that ESBL producing bacteria is present in different locations in dairy farms, and may be responsible for IMI. The detection of ESBLs on dairy herds could be a major concern for both public and animal health.

Electrolocation-communication discharges of the fish Gymnotus carapo L. (Gymnotidae: Gymnotiformes) during behavioral sleep

Technical problems have hampered the study of sleep in teleosts. The electrical discharges of Gymnotus carapo L. (Gymnotidae: Gymnotiformes) were monitored to evaluate their ease and reliability as parameters to study sleep. The discharges were detected by electrodes immersed in a glass aquarium and were recorded on a conventional polygraph. G. carapo showed conspicuous signs of behavioral sleep. During these periods, opercular beat rates were counted, electric discharges recorded, and the "sharp discharge increase" (SDI) of the orienting reflex was investigated. All 20 animals monitored maintained electrical discharges during behavioral sleep. The discharge frequencies during sleep (50.3 ± 10.4 Hz) were not significantly different from those observed when the fish was awake and inactive (57.2 ± 12.1 Hz) (Wilcoxon matched-pairs signed-ranks test, P>0.05). However, the SDI, which was prevalent in the awake fish, was not observed during periods of behavioral sleep. Additional observations showed that the species had cannibalistic habits. When presented with electrical discharges from a conspecific, the sleeping fish showed an initial decrease or pause in discharge frequency, while the awake fish did not have this response. We conclude that the electrical discharges of G. carapo were not conspicuous indicators of behavioral sleep. Discharges may have been maintained during sleep for sensory purposes, i.e., conspecific detection and avoidance of cannibalistic attacks.

Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins

Intercellular communication may be regulated by the differential expression of subunit gap junction proteins (connexins) which form channels with differing gating and permeability properties. Endothelial cells express three different connexins (connexin37, connexin40, and connexin43) in vivo. To study the differential regulation of expression and synthesis of connexin37 and connexin43, we used cultured bovine aortic endothelial cells which contain these two connexins in vitro. RNA blots demonstrated discordant expression of these two connexins during growth to confluency. RNA blots and immunoblots showed that levels of these connexins were modulated by treatment of cultures with transforming growth factor-ß1. To examine the potential ability of these connexins to form heteromeric channels (containing different connexins within the same hemi-channel), we stably transfected connexin43-containing normal rat kidney (NRK) cells with connexin37 or connexin40. In the transfected cells, both connexin proteins were abundantly produced and localized in identical distributions as detected by immunofluorescence. Double whole-cell patch-clamp studies showed that co-expressing cells exhibited unitary channel conductances and gating characteristics that could not be explained by hemi-channels formed of either connexin alone. These observations suggest that these connexins can readily mix with connexin43 to form heteromeric channels and that the intercellular communication between cells is determined not only by the properties of individual connexins, but also by the interactions of those connexins to form heteromeric channels with novel properties. Furthermore, modulation of levels of the co-expressed connexins during cell proliferation or by cytokines may alter the relative abundance of different heteromeric combinations.

Gap junctions in isolated rat aorta: evidence for contractile responses that exhibit a differential dependence on intercellular communication

Connexin43 (Cx43) is a major gap junction protein present in the Fischer-344 rat aorta. Previous studies have identified conditions under which selective disruption of intercellular communication with heptanol caused a significant, readily reversible and time-dependent diminution in the magnitude of a1-adrenergic contractions in isolated rat aorta. These observations have indentified a significant role for gap junctions in modulating vascular smooth muscle tone. The goal of these steady-state studies was to utilize isolated rat aortic rings to further evaluate the contribution of intercellular junctions to contractions elicited by cellular activation in response to several other vascular spasmogens. The effects of heptanol were examined (0.2-2.0 mM) on equivalent submaximal (»75% of the phenylephrine maximum) aortic contractions elicited by 5-hydroxytryptamine (5-HT; 1-2 µM), prostaglandin F2a (PGF2a; 1 µM) and endothelin-1 (ET-1; 20 nM). Statistical analysis revealed that 200 µM and 500 µM heptanol diminished the maximal amplitude of the steady-state contractile responses for 5-HT from a control response of 75 ± 6% (N = 26 rings) to 57 ± 7% (N = 26 rings) and 34.9 ± 6% (N = 13 rings), respectively (P<0.05), and for PGF2a from a control response of 75 ± 10% (N = 16 rings) to 52 ± 8% (N = 19 rings) and 25.9 ± 6% (N = 18 rings), respectively (P<0.05). In contrast, 200 µM and 500 µM heptanol had no detectable effect on the magnitude of ET-1-induced contractile responses, which were 76 ± 5.0% for the control response (N = 38 rings), 59 ± 6.0% in the presence of 200 µM heptanol (N = 17 rings), and 70 ± 6.0% in the presence of 500 µM heptanol (N = 23 rings) (P<0.13). Increasing the heptanol concentration to 1 mM was associated with a significant decrease in the magnitude of the steady-state ET-1-induced contractile response to 32 ± 5% (21 rings; P<0.01); further increasing the heptanol concentration to 2 mM had no additional effect. In rat aorta then, junctional modulation of tissue contractility appears to be agonist-dependent.

One hundred million years of interhemispheric communication: the history of the corpus callosum

Analysis of regional corpus callosum fiber composition reveals that callosal regions connecting primary and secondary sensory areas tend to have higher proportions of coarse-diameter, highly myelinated fibers than callosal regions connecting so-called higher-order areas. This suggests that in primary/secondary sensory areas there are strong timing constraints for interhemispheric communication, which may be related to the process of midline fusion of the two sensory hemifields across the hemispheres. We postulate that the evolutionary origin of the corpus callosum in placental mammals is related to the mechanism of midline fusion in the sensory cortices, which only in mammals receive a topographically organized representation of the sensory surfaces. The early corpus callosum may have also served as a substrate for growth of fibers connecting higher-order areas, which possibly participated in the propagation of neuronal ensembles of synchronized activity between the hemispheres. However, as brains became much larger, the increasingly longer interhemispheric distance may have worked as a constraint for efficient callosal transmission. Callosal fiber composition tends to be quite uniform across species with different brain sizes, suggesting that the delay in callosal transmission is longer in bigger brains. There is only a small subset of large-diameter callosal fibers whose size increases with increasing interhemispheric distance. These limitations in interhemispheric connectivity may have favored the development of brain lateralization in some species like humans. "...if the currently received statements are correct, the appearance of the corpus callosum in the placental mammals is the greatest and most sudden modification exhibited by the brain in the whole series of vertebrated animals..." T.H. Huxley (1).

Is avoidant disorder part of the social phobia spectrum in a referred sample of Brazilian children and adolescents?

The diagnosis of avoidant disorder was deleted from the Diagnostic and Statistical Manual of Mental disorders - fourth edition (DSM-IV) based on a `committee decision' suggesting that avoidant disorder is part of the social phobia spectrum. The objective of the present study was to examine the nature of this clinical association in a referred sample of Brazilian children and adolescents. We assessed a referred sample of 375 youths using semi-structured diagnostic interview methodology. Demographic (age at admission to the study and sex) and clinical (level of impairment, age at onset of symptoms and pattern of comorbidity) data were assessed in subsamples of children with avoidant disorder (N = 7), social phobia (N = 26), and comorbidity between both disorders (N = 24). Although a significant difference in the male/female ratio was detected among groups (P = 0.03), none of the other clinical variables differed significantly among subjects that presented each condition separately or in combination. Most of the children with avoidant disorder fulfilled criteria for social phobia. Thus, our findings support the validity of the conceptualization of avoidant disorder as part of the social phobia spectrum in a clinical sample.

Visual communication stimulates reproduction in Nile tilapia, Oreochromis niloticus (L.)

Reproductive fish behavior is affected by male-female interactions that stimulate physiological responses such as hormonal release and gonad development. During male-female interactions, visual and chemical communication can modulate fish reproduction. The aim of the present study was to test the effect of visual and chemical male-female interaction on the gonad development and reproductive behavior of the cichlid fish Nile tilapia, Oreochromis niloticus (L.). Fifty-six pairs were studied after being maintained for 5 days under one of the four conditions (N = 14 for each condition): 1) visual contact (V); 2) chemical contact (Ch); 3) chemical and visual contact (Ch+V); 4) no sensory contact (Iso) - males and females isolated. We compared the reproductive behavior (nesting, courtship and spawning) and gonadosomatic index (GSI) of pairs of fish under all four conditions. Visual communication enhanced the frequency of courtship in males (mean ± SEM; V: 24.79 ± 3.30, Ch+V: 20.74 ± 3.09, Ch: 0.1 ± 0.07, Iso: 4.68 ± 1.26 events/30 min; P < 0.05, two-way ANOVA with LSD post hoc test), induced spawning in females (3 spawning in V and also 3 in Ch+V condition), and increased GSI in males (mean ± SEM; V: 1.39 ± 0.08, Ch+V: 1.21 ± 0.08, Ch: 1.04 ± 0.07, Iso: 0.82 ± 0.07%; P < 0.05, two-way ANOVA with LSD post hoc test). Chemical communication did not affect the reproductive behavior of pairs nor did it enhance the effects of visual contact. Therefore, male-female visual communication is an effective cue, which stimulates reproduction among pairs of Nile tilapia.

Spectrum of K ras mutations in Pakistani colorectal cancer patients

The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients.

Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation.