The regeneration of the ventral nerve cord ganglion and peripheral tissues was investigated by radioimmunoassay and immunohistochemistry in the model animal, Eisenia fetida (Annelida, Oligochaeta). It is now well-established that pituitary adenylate cyclase-activating polypeptide (PACAP) is a neurotrophic factor, playing important roles in the development of the nervous system in vertebrate animals. Based on the apparent evolutionary conservation of PACAP and on the several common mechanisms of vertebrate and invertebrate nervous regeneration, the question was raised whether PACAP has any role in the regeneration of the earthworm nervous system. As a first step, we studied the distribution, concentration, and time-course of PACAP-like immunoreactivity during caudal regeneration of both lost segments and the ventral nerve cord ganglia in E. fetida. A strong upregulation of PACAP-like immunoreactivity was observed in most tissues following injury as determined by radioimmunoassay and immunohistochemistry. Significant increases in the concentration of PACAP-like compounds were found in the body wall, alimentary canal, and in coelomocytes. The most characteristic morphological feature was the accumulation of immunolabeled neoblasts in the injured tissues, especially in the ventral nerve cord ganglion that initiates and mediates regeneration processes. Our present results show that PACAP/PACAP-like peptides accumulate in the regenerating tissues of the earthworm, suggesting trophic functions of these compounds in earthworm tissues similarly to vertebrate species.

Nuclear estrogen receptor (ER), progesterone receptor (PR) and Ki-67 protein positive tumor cell fractions are semiquantitatively assessed in breast cancer for prognostic and predictive purposes. These biomarkers are usually revealed using immunoperoxidase methods resulting in diverse signal intensity and frequent inhomogeneity in tumor cell nuclei, which are routinely scored and interpreted by a pathologist during conventional light-microscopic examination. In the last decade digital pathology-based whole slide scanning and image analysis algorithms have shown tremendous development to support pathologists in this diagnostic process, which can directly influence patient selection for targeted- and chemotherapy. We have developed an image analysis algorithm optimized for whole slide quantification of nuclear immunostaining signals of ER, PR, and Ki-67 proteins in breast cancers. In this study, we tested the consistency and reliability of this system both in a series of brightfield and DAPI stained fluorescent samples. Our method allows the separation of overlapping cells and signals, reliable detection of vesicular nuclei and background compensation, especially in FISH stained slides. Detection accuracy and the processing speeds were validated in routinely immunostained breast cancer samples of varying reaction intensities and image qualities. Our technique supported automated nuclear signal detection with excellent efficacy: Precision Rate/Positive Predictive Value was 90.23 ± 4.29%, while Recall Rate/Sensitivity was 88.23 ± 4.84%. These factors and average counting speed of our algorithm were compared with two other open source applications (QuPath and CellProfiler) and resulted in 6-7% higher Recall Rate, while 4- to 30-fold higher processing speed. In conclusion, our image analysis algorithm can reliably detect and count nuclear signals in digital whole slides or any selected large areas i.e. hot spots, thus can support pathologists in assessing clinically important nuclear biomarkers with less intra- and interlaboratory bias inherent of empirical scoring. © 2017 International Society for Advancement of Cytometry.

The anatomical organization of the peripheral and central sensory structures of the earthworm Lumbricus terrestris was investigated applying a fluorescent carbocyanine dye (DiI) as a neuronal tracer. Using whole-mount preparations and confocal laser scanning microscopy, the pattern of primary sensory cells and pathways of their processes were traced and reconstructed in three-dimensions. Our study shows that a ventral nerve cord ganglion receives sensory fibers from at least two adjacent segments suggesting that the peripheral nervous system is not segmental in its arrangement and the receptive-fields of the body wall overlap in earthworms. Furthermore, our result suggests an integrative function of the basiepidermal plexus consists of sensory and motor fibers.

By means of radioimmunoassay, we studied the concentration of pituitary adenylate cyclase-activating polypeptide (PACAP)-like proteins in intact and regenerating earthworms. Transection of animals increased the concentration of PACAP-like compounds in coelomocytes, and a decreasing rostrocaudal gradient was detected in the regenerating animals. Western blot analysis revealed a range of PAC1-receptor proteins with molecular weights from 40 to 80 kDa. Electron microscopic immunocytochemistry showed that PAC1 receptors were located on distinct sets of coelomocytes (mainly on amebocytes and on some granulocytes). Based on our results we hypothesize a link between PACAP and coelomocytes, suggesting that PACAP modulates the function of amebocytes and certain granulocytes that play a role in tissue remodeling of regenerating earthworms.

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