Magnus Hansson

Immunoreactive atrial and brain natriuretic peptides are co-localized in Purkinje fibres but not in the innervation of the bovine heart conduction system

SummaryRecently, we observed that atrial natriuretic peptide (ANP) immunoreactivity was present in Purkinje fibres and nerve fibre varicosities in the conduction system of the bovine heart. In order to elucidate further the morphological correlation between natriuretic peptides and the conduction system, the distribution of brain natriuretic peptide (BNP) was examined. The different parts of the conduction system in the bovine heart were dissected out and processed for immunohistochemistry with antisera against BNP and ANP. BNP immunoreactivity was frequently observed in Purkinje fibres of the atrioventricular bundle, whereas only a few Purkinje fibres in the ventricular part of the conduction system showed immunoreaction. BNP immunoreactivity was detected in regions of the Purkinje fibres which also showed ANP immunoreactivity. BNP immunoreactivity was not observed in nerve fibre varicosities. Methodologically, a larger number of small BNP immunofluorescent granular structures was observed by using an elution-restaining technique instead of conventional immunohistochemistry. The present study shows that BNP and ANP immunoreactivities frequently occur in the atrioventricular bundle and that they are co-localized in Purkinje fibres, but not in nerve fibre varicosities, in the conduction system. As previously has been proposed for ANP, the present observations suggest that also BNP may act in an autocrine and/or paracrine way in the conduction cells.

Embolic material generated by multiple aortic crossclamping: a perfusion model with human cadaveric aorta

BACKGROUND Atherosclerosis of the ascending aorta and use of aortic crossclamping are risk factors for neurologic injury during cardiac surgery. OBJECTIVES Repeated aortic manipulation is part of the surgical approach to most cardiac operations. The aim of this study was to assess the amount and size of particulate matter that is dislodged from the aortic wall as a function of repeated aortic crossclamping. METHODS In 10 subjects undergoing autopsy the aorta was dissected and mounted in a perfusion model. The ascending aorta was crossclamped and washed out 10 times, with the perfusate collected in aliquots (1 to 10). The aliquots were examined by computerized image processing, both macroscopically and under the microscope for calcified and cellular material. RESULTS Aortic crossclamping produced substantial output of particulate matter. After repeated aortic crossclamping the number of particles decreased (P =.012) and approached the baseline for aliquots 6 to 10. The average particle diameter was 0.63 +/- 0.03 mm, with a maximum of 4.74 mm. Similar variability in particle outputs were recorded microscopically, with findings of both calcified and cellular material. Nine of 10 aortas had calcifications seen during simple visual inspection. CONCLUSIONS The washouts of dislodge material at aortic crossclamping had embolic potential. During the initial aortic crossclamping procedures the amount of particles was substantial, both macroscopically and microscopically. On the microscopic scale noncalcified cellular debris represents a significant pool of embolic material. Repeated aortic crossclamping reduced the amount of particles. These findings question surgical techniques associated with repeated aortic crossclamping.

Presence of immunoreactive atrial natriuretic peptide in nerve fibres and conduction cells in the conduction system of the bovine heart.

Previous findings of atrial (A-type) natriuretic peptide (ANP) in nervous tissue, such as the brain and the superior cervical ganglia, led us to investigate the possible occurrence of ANP in nervous tissue in the heart. The distribution of ANP in the bovine heart, particularly its conduction system, was examined by the use of immunohistochemical methods and an antiserum against α-hANP. ANP immunoreactivity was frequently detected in atrial myocytes and in the Purkinje fibres of the AV-bundle, and was sometimes seen in the Purkinje fibres of the bundle branches and their ramifications. On the other hand, ANP immunoreactivity was never seen in the conduction cells of SA- and AV-nodes. ANP immunoreactivity was also detected in small nerve-fibre varicosities, mainly in the AV-node and AV-bundle. Most of these varicosities were located in the proximity of the conduction cells, but some occurred close to fine blood vessels or in the walls of arterioles. These observations show for the first time that ANP immunoreactivity is present not only in atrial myocytes and conduction cells but also in nerve-fibre varicosities in the conduction system. The observations suggest that ANP may act as a neuromodulator and/or neurotransmitter in the conduction system.

Natriuretic peptide immunoreactivity in nerve structures and Purkinje fibres of human, pig and sheep hearts

Atrial natriuretic peptide is a well-described peptide in cardiac Purkinje fibres and has been shown to interfere with the autonomic regulation in the heart of various species, including man. Recently, we detected immunoreactivity for the peptide in intracardial ganglionic cells and nerve fibre varicosities of bovine hearts, by the use of a modified immunostaining technique that induced an improved detection of natriuretic peptides. These findings raised the question as to whether natriuretic peptides are detectable in these tissues in man and other species. The conduction system from human, pig and sheep hearts was dissected and processed with antisera against atrial natriuretic peptide and the closely related brain natriuretic peptide. Immunostaining for the brain natriuretic peptide was detected in some Purkinje fibres in all of these species. Interestingly, in pig, sheep and human hearts, some ganglionic cells and nerve fibres showed atrial natriuretic peptide immunoreactivity, particularly in the soma of human ganglionic cells. This is the first study showing immunoreactivity for the atrial natriuretic peptide in nerve structures and for the brain natriuretic peptide in Purkinje fibres of the human heart. The results give a morphological correlate for the documented effects of atrial natriuretic peptide on the heart autonomic nervous system and for the presumable effects of brain natriuretic peptide in the conduction system of man

YWHAE-FAM22 gene fusion in clear cell sarcoma of the kidney.

We read with great interest the recent publication in J Pathol by O’Meara et al, describing the identification of the target genes of the t(10;17)(q22;p13) translocation in clear cell sarcoma of the kidney (CCSK) [1]. In this excellent paper the authors show that the t(10;17) translocation in CCSK results in a YWHAE–FAM22 gene fusion in which exons 1–5 of YWHAE are linked to exons 2–7 of FAM22. The fusion was detected by FISH and/or RT–PCR in a previously published CCSK with a t(10;17) translocation, as well as in six of 50 further CCSKs tested (overall frequency 12%). Fusionpositive CCSKs were shown to have a significantly higher tumour cellularity compared to fusion-negative tumours (p < 0.001). CCSK is an aggressive paediatric renal cancer preferentially affecting young males [2]. The tumour is defined based on its typical light microscopic appearance, which deviates from that of mesoblastic nephroma and Wilms’ tumour. CCSK also shows a striking propensity to metastasize to bone, as opposed to Wilms’ tumour, which classically spreads to lymph nodes, lung and liver [2]. Of particular difficulty in the diagnosis of CCSK is the pronounced morphological diversity that these tumours may show, ranging from epithelioid to spindle-cell growth patterns, mimicking certain subtypes of Wilms’ tumour. Therefore, there is a need for diagnostically useful biomarkers which may assist in accurately identifying CCSK. As part of a comprehensive screen for tumourtype specific chromosome translocations in sarcomas at our institute, we previously identified a rare type of sarcoma in a 3 week-old boy with a large congenital tumour involving the soft tissues of the right neck (previously unpublished case). The clinicopathological characteristics of this case (designated case 1) are summarized in Table 1. Imaging studies revealed that the lesion extended from the foramen ovale near the cerebellum to the thoracic inlet. A fine needle aspirate was performed, followed by an incisional biopsy. Histopathologically, the tumour showed dense cellularity with uniform, small, primitive cells with poorly defined, lightly staining or clear cytoplasm (Figure 1A). The nuclei were round vesicular with a few small nucleoli. There were solid sheets of tumour cells in focal trabecular and multinodular arrangements. The tumour cells were intimately associated with a prominent vasculature resembling capillaries and venule-like channels. There were numerous mitoses and small foci of necrosis and vesicular invasion. Immunophenotypically, the tumour cells stained positive for vimentin and were negative for cytokeratins, LCA, HBA71 (CD99), NSE, muscle-specific actin (HHF35), S-100 protein, HMB45, desmin, α-smooth muscle actin, α-fetoprotein, chromogranin A, L26 (CD20), and UCHL1 (data not shown). The histopathology and immunoprofile were consistent with the diagnosis of an extrarenal CCSK [3,4]. The tumour did not respond to chemotherapy and the patient developed brain metastases 6 months later and died a few days thereafter. Cytogenetic analysis of the incisional biopsy from case 1 revealed a pseudodiploid male karyotype with two balanced translocations as the sole anomalies, t(10;17)(q22;p13) and t(8;11)(q22;p13) (Figure 1B). FISH analysis using painting probes confirmed the t(8;11) and t(10;17) translocations (data not shown). This is, to our knowledge, the first report of an extrarenal CCSK with a t(10;17) translocation and the fourth case of CCSK reported to date with this rearrangement. Inspired by the interesting findings of O’Meara and co-workers [1], we analysed our t(10;17)-positive CCSK for expression of the YWHAE–FAM22 gene fusion. Total RNA was isolated from frozen tumour cells and converted to cDNA as previously described [5]. RT–PCR analysis using previously described primer sequences [1] revealed expression of a 236 bp fragment (Figure 1C) which, after nucleotide sequence analysis, was shown to correspond to a YWHAE– FAM22 fusion transcript (Figure 1D) identical to that reported by O’Meara et al. [1]. Of interest, in agreement with O’Meara et al, this fusion-positive case was also a highly cellular CCSK at a late stage of the disease. We also searched the files of the Gothenburg Musculo-skeletal Tumour Centre for CCSKs and identifed three additional cases during the last 4 years. The samples were obtained with Institutional Review Board approval. The clinicopathological characteristics of these three cases are summarized in Table 1. Total RNA was extracted from five 10 μm sections obtained from paraffin blocks of each of these three cases and converted to cDNA as previously described [5]. Screening of these cDNAs for expression of YWHAE–FAM22 fusion transcripts, using primer sequences optimized for amplification of shorter transcript fragments in formalin-fixed paraffin-embedded material [1], revealed that all three tumours were negative for the YWHAE–FAM22 fusion (Figure 1C). Thus, one out of four (25%) of our CCSKs were fusion-positive, confirming that the YWHAE–FAM22 fusion is indeed a biomarker that identifies a subset of CCSK. In an effort to identify additional genetic alterations that may cooperate with the YWHAE–FAM22 fusion or characterize fusion-negative CCSKs, we also isolated DNA from three of our four cases of CCSK (there was no material available from case 2) and performed highresolution array CGH analyses using 244K arrays, as previously described [6]. Detailed analysis of the arrays

Ingrowth of sympathetic innervation occurs concomitantly with a decrease of ANP in the growing rat cardiac ventricles.

The relationship between the immunohistochemical expression of atrial natriuretic peptide and the occurrence of sympathetic nerve fibres, as visualized by staining for tyrosine hydroxylase, in the growing rat heart was evaluated. Rats were investigated at four different stages from birth to 21 days postnatally. The effects of chemical destruction of sympathetic nerve terminals in neonatal rats on the cardiac atrial natriuretic peptide content were furthermore examined by use of radioimmunoassay. There was in principle a reciprocal pattern of immunoreaction for atrial natriuretic peptide and tyrosine hydroxylase positive innervation in the ventricular myocardium, atrial natriuretic peptide reaction becoming less and less pronounced with the ingrowth of innervation positive for tyrosine hydroxylase. Furthermore, in the peripheral Purkinje fibre network, there was a marked atrial natriuretic peptide immunoexpression and scarce or no nerve fibres throughout the examination period. The radioimmunoassay measurements showed that chemical sympathectomy lead to elevated cardiac levels of atrial natriuretic peptide. The study shows that sympathetic innervation grows into the ventricular parts concomitantly with the occurrence of a decline in atrial natriuretic peptide expression during development of the heart. Furthermore, it is shown that a reversion of the in growth of sympathetic innervation by destruction of cardiac sympathetic nerves at an early stage leads to increased levels of atrial natriuretic peptide in the heart. The results give new evidence to the phenomenon that the atrial natriuretic peptide levels in the ventricular myocardium and the peripheral parts of the conduction system are under influence of the presence of sympathetic innervation.

Atrial natriuretic peptide in the innervation of the bovine heart conduction system: relationship with substance P and autonomic innervation - immunohistochemical studies

Recently, we observed that atrial natriuretic peptide (ANP) immunoreactivity (IR) was present not only in the Purkinje fibres, but also in nerve fibre varicosities in the conduction system of the bovine heart. These findings and previous observations that ANP is able to influence autonomic neurotransmission in the heart, lead us to elucidate the possible occurrence of ANP in the sympathetic and/or parasympathetic nervous systems and/or in various types of peptidergic innervation in the conduction system. The different parts of the conduction system of bovine hearts were dissected out and processed for immunohistochemistry including double-staining, using antisera against ANP, tyrosine hydroxylase and different neuropeptides. We observed that some of the nerve fibre varicosities exhibiting ANP-IR showed substance P-IR and that ANP was present as scattered immunoreactive granules in intracardial, presumably parasympathetic, ganglionic cells. The study shows that ANP is likely to be present in parasympathetic innervation and in afferent nerve endings in the bovine heart conduction system.

Decrease in binding for the neuropeptide VIP in response to marked inflammation of the mucosa in ulcerative colitis

Abstract:  The neuropeptide vasoactive intestinal peptide (VIP) is involved in the neuroimmunomodulation of the intestine. In the present study, specimens from the sigmoid colon of ulcerative colitis (UC) and non‐UC patients were examined for immunohistochemistry and in vitro receptor autoradiography. Marked occurrence of VIP binding was observed in the mucosa. However, there were very low levels of binding in areas showing pronounced inflammation/derangement. The study shows that marked derangement of the mucosa leads to a distinct decrease in VIP binding. Thus, it is possible that a decrease in trophic and anti‐inflammatory VIP effects occurs in areas exhibiting a very marked inflammation.

Primary spinal intradural mesenchymal chondrosarcoma with detection of fusion gene HEY1‑NCOA2: A paediatric case report and review of the literature

Mesenchymal chondrosarcoma is an extremely rare malignant tumour that most commonly originates in the bone, but is also present in extraskeletal sites. The tumour is morphologically characterized by a biphasic pattern of small round cells and islands of cartilage. Spinal mesenchymal chondrosarcomas are even rarer and, therefore, few investigations exist regarding the biological behaviour of the tumours. In the present study, we report a case of a 10-year-old female presenting with 9 months of back pain and radiographic findings of an intradural lesion measuring 1.5 cm at the level of Th4. The tumour was completely excised and subjected to pathological analyses. Following detection of the HEY1-NCOA2 fusion gene, the tumour was morphologically and immunohistochemically defined as an intradural mesenchymal chondrosarcoma attached to the dura mater. In this study, we validate the recent identification of the fusion gene HEY1-NCOA2 in paediatric extraskeletal mesenchymal chondrosarcomas. The relevant literature is reviewed and further discussed in relation to our findings.

Binding Sites for VIP in the Reorganizing Mucosa of the Irradiated Bowela

Abstract: Rats were given radiotherapy (total dose 30 Gy) over the abdomen. Seven days later specimens of the duodenum were prepared for in vitro receptor autoradiography using the radioligand [125I]VIP. The autoradiograms were quantitatively analyzed using a computer system. Histological examination revealed that a very marked reorganization of the mucosa had occurred in response to irradiation. Using receptor autoradiography, we found [125I]VIP‐specific binding sites in the reorganizing mucosa, except where denudation had occurred. Such binding sites also occurred in the smooth muscle layer of the duodenal wall. The observations suggest that VIP has profound effects in radiation‐induced enteropathy.