Indirect Immunoperoxidase Technique Research Articles

Cell Death Mechanism of Organometallic Ruthenium(II) and Iridium(III) Arene Complexes on HepG2 and Vero Cells.

Due to side effects and toxicity associated with platinum-derived metal-based drugs, extensive research has been conducted on ruthenium (Ru) complexes. We aim to synthesize a highly oil soluble Ru(II)-p-cymene complex (Ru1) with an aliphatic chain group, a bimetallic Ru(II)-p-cymene complex (Ru2) with N,S,S triple-coordination and a bimetallic Ir(III)-pentamethylcyclopentadienyl complex (Ir1) with S,S double-coordination. Subsequently, we investigate the effects of these complexes on Vero and HepG2 cell lines, focusing on cell death mechanisms. Characterization of the complexes is performed through nuclear magnetic resonance spectroscopy (1H and 13C NMR) and Fourier-transform infrared spectroscopy. The effective doses are determined using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT) assay, applying different doses of the complexes to the two cell lines for 24 and 48 h, respectively. Immunoreactivities of Bax, Bcl2, caspase-3, RIP3, and RIPK1 are analyzed using the indirect immunoperoxidase technique. Notably, all the complexes (Ru1, Ru2, and Ir1) exhibit distinct cell death mechanisms, showing greater effectiveness than cisplatin. This study reveals the diverse mechanisms of action of Ru and Ir complexes based on different ligands. To the best of our knowledge, this study represents the first investigation of a novel RAED-type complex (Ru1) and unexpected bimetallic complexes (Ru2 and Ir1).

OVER SERÖZ KARSİNOMUNDA VE BORDERLİNE SERÖZ TÜMÖRDE İMMÜNHİSTOKİMYASAL PINCH/LIMS-1 ANTİKOR EKSPRESYONUNUN KLİNİKOPATOLOJİK DEĞERİ

Objective
 Particularly interesting cys-his rich protein (PINCH/
 LIMS-1), a protein implicated in cell adhesion, is
 assumed to oversee the development and invasion of
 cancer cells in tumors and tumor-associated stroma.
 This study aimed to assess PINCH-1 expression in
 serous borderline tumor (SBT) and serous carcinoma
 (SC) in the tumor and peritumoral stroma and
 scrutinize any associations between its expression
 and various clinical and pathological parameters.
 Material and Method
 In this study, the expression of the PINCH-1 antibody
 was analyzed in 21 cases of SBT and 89 cases of
 SC using the indirect immunoperoxidase technique
 with streptavidin/HRP-biotin. The staining pattern of
 PINCH-1 in the tumor and peritumoral stroma was
 evaluated using a semiquantitative scoring method.
 The staining procedure used in the study allowed for
 the accurate identification of PINCH-1 expression,
 and the data obtained through the semiquantitative
 scoring method provided a reliable of assessing
 the degree and intensity of PINCH-1 staining. Thus,
 the correlation between PINCH-1 expression and
 various pathologic factors such as patient age, tumor
 size, FIGO stage, intra-abdominal washing cytology,
 capsule invasion, tumor location in the ovary, tumor
 grade, and cancer antigen 125 (CA125) levels at the
 time of diagnosis was examined.
 Results
 The study found that PINCH-1 was more prevalent
 in cases of SC than in SBT cases. The tumors in SC
 cases had stronger staining than those in SBT cases
 (p

Boron-Doped Carbon Nanodots as a Theranostic Agent for Colon Cancer Stem Cells.

Carbon nanodots have drawn a great deal of attention due to their green and expedient opportunities in biological and chemical sciences. Their high fluorescence capabilities and low toxicity for living cells and tissues make them excellent imaging agents. In addition, they have a fluorimetric response against inorganic and organic species. Boron-doped carbon nanodots (B-CDs) with high fluorescence yield were produced from phenylboronic acid and glutamine as boron and carbon sources, respectively, by a hydrothermal method. First, the effects of the temperature on their fluorescence yield and the structural characteristics of B-CDs were investigated. Second, their cytotoxicity and cell death and proliferation behaviors were examined. The cytotoxicity was evaluated by the MTT assay. The cellular properties were evaluated with the distribution of caspase 3, Ki67, lamin B1, P16, and cytochrome c after the indirect immunoperoxidase technique. After the MTT assay, 1:1 dilution of all applicants for 24 h was used in the study. After immunohistochemical analyses, the application of B-CDs synthesized at 230 °C did not change control cell (Vero) proliferation, and also apoptosis was not triggered. Colo 320 CD133+ and CD133- cell-triggered apoptosis and cellular senescence were found to be synthesis temperature dependent. In addition, Colo 320 CD133- cells were affected relatively more than CD133+ cells from B-CDs. While B-CDs did not affect the control cells, the colon cancer stem cells (Colo 320 CD133+) were affected in a time-dependent manner. Therefore, the use of the synthesized B-CD product may be an alternative method for controlling or eliminating cancer stem cells in the tumor tissue.

Иммунные реакции при лимфоме Ходжкина

HL is characterized by significantly enlarged lymph nodes and the presence of rare Hodgkin and Reed-Sternberg cells. Pathogenesis is not fully understood. The increase in the disease risk can be associated with immunosuppression, HIV, parenchymal organ transplantation, autoimmune disorders, etc. The possibility of differentiating pathogenetic and protective immune responses associated with this disease will help understand the causes of the disease and the treatment prognosis. The study was aimed to determine the features of immune responses in HL depending on the disease duration and the circulating lymphocyte counts. A total of 134 patients with HL were assessed. The cytogram and phagocytosis were assessed in blood smears stained by the Wright-Giemsa procedure. The expression of lymphocyte markers in lymphocytes was determined using the indirect immunoperoxidase technique and flow cytometry. Serum levels of cytokines, immunoglobulins, autoantibodies and circulating immune complexes were assessed by enzyme immunoassay. Comparative analysis of the immune responses depending on peripheral blood leukocyte counts is provided. It has been found that prolonged HL course is associated with the decrease in the functionally active T cell counts, progressive neutropenia and monocytopenia, along with the increased activity of the reaginic reactions and autosensitization. In individuals with lymphocytopenia, mainly small lymphocytes die, the 3-fold decrease in the counts of such lymphocytes is observed; lymphocytopenia is associated with the deficiency of circulating T cells, both mature and immature, the concentrations of which decrease by 2.5–3 times, while B cell counts show no dramatic changes. The disease progression is associated with reduction of the lymphocyte homeostasis control by granulocytes and monocytes, along with progressive neutropenia and monocytopenia.

Histologic and physiologic analysis of the relationship between the dorsal nerve of the penis and the corpus cavernosum on a rat model. A complementary pathway on the innervation of penile erection?

The dorsal nerve of the penis (DNP) is the terminal branch of the pudendal nerve which is responsible for the somatic innervation of the penis. This study aims to outline any direct role of the DNP in the hemodynamics of erection histologically and physiologically. Fifteen Wistar albino rats were sorted into the electrical activity (n = 6), intracavernous pressure (n = 4), and control (n = 5) groups. The dorsal nerve was electrostimulated and the simultaneous changes in intracavernous pressure and smooth muscle activity were recorded. Penile tissues were collected, fixed, and sectioned, the slides were stained with either hematoxylin-eosin for morphological evaluation or using the indirect immunoperoxidase technique to analyze the distributions of eNOS, iNOS, and nNOS. During electrostimulation, there was a simultaneous statistically significant decrease in the electrical activity inside the corpora in electromyography and an increase in intracavernous pressure. eNOS and iNOS immunoreactivities were higher in the study group than in the control group. nNOS immunoreactivity was moderate in both study and control groups. Some fibers in the dorsal nerve of penis continue into the corpora cavernosa through the tunica albuginea and have an active, direct role in the hemodynamic process of erection, which may be complementary to the main route of innervation.

CHARACTERISTICS OF SUBCLONAL STRUCTURE IN THYROCYTE POPULATION IN RADIOIODINE-REFRACTORY METASTASES OF PAPILLARY THYROID CANCER

The aim of the study was cytological and immunocytochemical researches of thyrocyte populations in fine-needle aspiration smears of radioiodine-refractory metastases and their comparison with radioiodine-avid metastases and corresponding primary papillary carcinoma of the thyroid. Materials and Methods. The cytomorphological and immunocytochemical researches were conducted on the fine needle aspiration smears of 60 papillary thyroid carcinomas and 104 metastases, which were detected in the postoperative period. We applied the indirect immunoperoxidase technique using primary monoclonal mouse antibodies against leukocyte-common antigen, macrophage antigen, thyroglobulin, epithelial cell adhesion molecule, cytokeratines 7.8, polyclonal rabbit antibodies against calcitonin. Results. It was demonstrated, that radioiodine-avid metastases and corresponding primary papillary thyroid carcinoma smears had first type of cellular population with a relatively regular location of thyrocytes. Unlike them, radioiodine-refractory metastases smears had the second type of cellular population with irregularly location of thyrocytes among which showed up two cellular phenotypes. In our investigated smears some special cellular complexes were found – in 21 % of radioiodine-refractory metastases, 1 % in radioiodine-avid metastases and none of it in corresponding primary papillary thyroid. The cytological sign of cystic degeneration was found in 58 % of radioiodine-refractory metastases, but in radioiodine-avid metastases – was absent. Conclusion. The radioiodine-refractory metastases of papillary thyroid cancer demonstrated their distinction from radioiodine-uptake metastases with a presence of phenotypic heterogeneity. It is shown, that an appearance of certain cell subpopulations, special cellular complexes and cystic degeneration in fine-needle aspiration smears of radioiodine-refractory metastases, which in radioiodine-uptake metastases and corresponding primary papillary thyroid carcinomas were not found, can be used during the preoperative period to forecast the impossibility of radioiodine treatment.

Isolation and identification of Bovine coronavirus (BCoV) from diarrheatic calves in Dakahlia Governorate, Egypt

In the current study, a total of 107 fecal samples were collected from diarrhetic calves from 1 day to 3 weeks of age from different localities in Dakahlia governorate, Egypt (Gamasa, Belkas, Sherbin, Nabaroh, Talkha, Almanzala, Algamalia, and Aga) during the period from year 2015 to year 2018. These calves were suffering from watery diarrhea, dehydration, weakness and recumbency. All samples were taken from calves which were born from vaccinated cows. The fecal samples from 107 cases were screened for the presence of BCoV by using Ag-ELISA and RT-PCR. From ELISA and PCR positive identified samples, we visualized the Coronavirus particles when negatively stained by transmission electron microscopy. From ELISA and PCR positive identified samples, virus isolation was done via MDBK tissue culture, six passages were carried out followed by identification of BCoV in harvested tissue culture via DFA test and indirect immunoperoxidase technique. Furthermore, nucleotide sequence analysis of amplified N gene of the identified BCoV nucleic acid was done and compared with published reference sequences on GeneBank. The results of ELISA and RT-PCR revealed that: out of 107 tested fecal samples, 4 samples showed positive results (3.7%) and 103 samples showed negative results (96.2%). The viral particles showed pleomorphism with characteristic radial projections forming a corona, by transmission electron microscopy. All tested samples gave positive results with DFAT as yellowish green coloration was detected in stained MDBK cells which increased gradually with increased passages. All tested samples gave positive results with IP as brownish coloration was detected in stained MDBK cells which increased gradually with increased the period of incubation. further studies is required for better understanding the genetic diversity among BCoV circulating in Egyptian farms based on full genome sequencing.

Isolation and identification of Bovine coronavirus (BCoV) from diarrheatic calves in Dakahlia Governorate, Egypt

In the current study, a total of 107 fecal samples were collected from diarrhetic calves from 1 day to 3 weeks of age from different localities in Dakahlia governorate, Egypt (Gamasa, Belkas, Sherbin, Nabaroh, Talkha, Almanzala, Algamalia, and Aga) during the period from year 2015 to year 2018. These calves were suffering from watery diarrhea, dehydration, weakness and recumbency. All samples were taken from calves which were born from vaccinated cows. The fecal samples from 107 cases were screened for the presence of BCoV by using Ag-ELISA and RT-PCR. from ELISA and PCR positive identified samples, we visualized the Coronavirus particles when negatively stained by transmission electron microscopy. from ELISA and PCR positive identified samples, virus isolation was done via MDBK tissue culture, six passages were carried out followed by identification of BCoV in harvested tissue culture via DFA test and indirect immunoperoxidase technique. Furthermore, nucleotide sequence analysis of amplified N gene of the identified BCoV nucleic acid was done and compared with published reference sequences on GeneBank. The results of ELISA and RT-PCR revealed that: out of 107 tested fecal samples, 4 samples showed positive results (3.7%) and 103 samples showed negative results (96.2%). The viral particles showed pleomorphism with CHARacteristic radial projections forming a corona, by transmission electron microscopy. All tested samples gave positive results with DFAT as yellowish green coloration was detected in stained MDBK cells which increased gradually with increased passages. All tested samples gave positive results with IP as brownish coloration was detected in stained MDBK cells which increased gradually with increased the period of incubation. further studies is required for better understanding the genetic diversity among BCoV circulating in Egyptian farms based on full genome sequencing.

Keratinocytes derived from embryonic stem cells induce wound healing in mice

ABSTRACTThe skin plays an important role in defending the body against the environment. Treatments for burns and skin injuries that use autologous or allogenic skin grafts derived from adult or embryonic stem cells are promising. Embryonic stem cells are candidates for regenerative and reparative medicine. We investigated the utility of keratinocyte-like cells, which are differentiated from mouse embryonic stem cells, for wound healing using a mouse surgical wound model. Mice were allocated to the following groups: experimental, in which dressing and differentiated cells were applied after the surgical wound was created; control, in which only the surgical wound was created; sham, in which only the dressing was applied after the surgical wound was created; and untreated animal controls with healthy skin. Biopsies were taken from each group on days 3, 5 and 7 after cell transfer. Samples were fixed in formalin, then stained with Masson’s trichrome and primary antibodies to interleukin-8 (IL-8), fibroblast growth factor-2 (FGF-2), monocyte chemoattractant protein-1 (MCP-1), collagen-1 and epidermal growth factor (EGF) using the indirect immunoperoxidase technique for light microscopy. Wound healing was faster in the experimental group compared to the sham and control groups. The experimental group exhibited increased expression of IL-8, FGF-2 and MCP-1 during early stages of wound healing (inflammation) and collagen-1 and EGF expression during late stages of wound healing (proliferation and remodeling). Keratinocytes derived from embryonic stem cells improved wound healing and influenced the wound healing stages.

Comparative diagnosis of Mycobacterium avium subspecies paratuberculosis in the tissues of clinical and subclinical sheep of paratuberculosis endemic farm

Paratuberculosis (Johne’s disease) is the chronic infectious granulomatous enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis. Due to the lack of definite diagnostic tests, the early detection of the disease is difficult. The present study was undertaken to assess comparative efficacy of diagnostic methods routinely used in the identification and confirmation of Mycobacterium avium subspecies paratuberculosis (MAP) in the tissue samples of naturally infected clinical and subclinical sheep. The ileum and mesenteric lymph node (MLN) tissues were collected from 20 paratuberculous sheep from organized farm of Rajasthan. These animals were further classified as paucibacillary (PB) (n = 8) or multibacillary (MB) (n = 12) on the basis of histopathological findings and mycobacterial loads. The ileum and MLN sections from PB and MB and uninfected control groups were tested by indirect immunoperoxidase technique (IPT), Ziehl Neelsen’s (ZN) method, bacterial culture, IS900 PCR and 251 gene PCR. In PB sheep, IPT and ZN were positive in 87.5 and 50% of intestinal sections and 75 and 37.5% of MLN sections, respectively. In MB sheep, IPT and ZN had equal sensitivity. The overall sensitivity of IPT was found to be superior (95%) than ZN method (80%) in demonstration of acid-fast bacteria in intestinal tissues. On the bacterial cultural examination of the intestinal and MLN tissues using Herrold’s egg yolk medium, MAP was isolated in eight (66.6%) MB and in four (50%) PB sheep. Among 20 sheep, IS900 PCR detected 13 (65.0%) and 251 gene PCR detected 16 (80%) sheep as positive for MAP. In the PB sheep, the culture, ZN test and PCR sensitivity was found low in comparison to MB sheep. The sensitivity of IPT in PB sheep was better than other tests in detecting the MAP infection, which underlines its utility in confirmation of subclinical and clinical cases of paratuberculosis in sheep. The sensitivity of 251 gene PCR assay was found better than IS900 gene PCR and has potential to be used as screening test for clinical and subclinical paratuberculosis in sheep flocks.

Periostin expression in neoplastic and non-neoplastic diseases of bone and joint

BackgroundPeriostin is a matricellular protein that is expressed in bone and joint tissues. To determine the expression of periostin in primary bone tumours and to assess whether it plays a role in tumour progression, we carried out immunohistochemistry and ELISA for periostin in a range of neoplastic and non-neoplastic bone and joint lesions.Methods140 formalin-fixed paraffin-embedded sections of bone tumours and tumour-like lesions were stained by an indirect immunoperoxidase technique with a polyclonal anti-periostin antibody. Periostin expression was also assessed in rheumatoid arthritis (RA) and non-inflammatory osteoarthritis (OA) synovium and synovial fluid immunohistochemistry and ELISA respectively.ResultsPeriostin was most strongly expressed in osteoid/woven bone of neoplastic and non-neoplastic bone-forming lesions, including osteoblastoma, osteosarcoma, fibrous dysplasia, osteofibrous dysplasia, fracture callus and myositis ossificans, and mineralised chondroid matrix/woven bone in chondroblastoma and clear cell chondrosarcoma. Reactive host bone at the edge of growing tumours, particularly in areas of increased vascularity and fibrosis, also stained strongly for periostin. Vascular elements in RA synovium strongly expressed periostin, and synovial fluid levels of periostin were higher in RA than OA.ConclusionsIn keeping with its known role in modulating the synthesis of collagen and other extracellular matrix proteins in bone, strong periostin expression was noted in benign and malignant lesions forming an osteoid or osteoid-like matrix. Periostin was also noted in other bone tumours and was found in areas of reactive bone and increased vascularity at the edge of growing tumours, consistent with its involvement in tissue remodelling and angiogenesis associated with tumour progression.

Identification of Bulge Stem Cells in Mouse and Human Hair Follicles

The skin contains various populaions of stem cells, but its characterization has been hampered by lack of markers and unclear location. The hair follicle has a niche for stem cells called a “bulge” which acts as a reservoir of multipotent stem cells. In the study reported here, an immunohistochemical and immunofluorescence analysis was performed on mouse and human tissues in order to determine the possible presence of stem cells of hair follicle through cytokeratin 15 (CK15), CD34, and CD200 markers identified as crucial to the stem cells and to identify the bulge region. Mouse (n = 7) and human (n = 7) skin samples were used. The expression of proteins was determined by the indirect immunoperoxidase technique and a secondary antibody bound to a fluorochrome. The specificity of staining was evaluated by negative controls. The results revealed that the stem cells associated with CD34 and CD200 antibodies were differentially expressed in the interfollicular epidermis, sebaceous glands, and bulge region, indicating that, in mice, CD34 and, in humans, CD200 are more specific than CK15 in detecting bulge cells. It also suggests that CD34 is specific for mouse bulge cells, while CD200 might have specificity for progenitor cells and partially differentiated cells in humans.

Detection of pantothenic acid-immunoreactive neurons in the rat lateral septal nucleus by a newly developed antibody.

The available immunohistochemical techniques have documented restricted distribution of vitamins in the mammalian brain. The aim of the study was to develop a highly specific antiserum directed against pantothenic acid to explore the presence of this vitamin in the mammalian brain. According to ELISA tests, the anti-pantothenic acid antiserum used showed a good affinity (10-8 M) and specificity. The antiserum was raised in rabbits. Using an indirect immunoperoxidase technique, the mapping of pantothenic acid-immunoreactive structures was carried out in the rat brain. Pantothenic acid-immunoreactive perikarya were exclusively found in the intermediate part of the lateral septal nucleus. These cells were generally small, round, fusiform or pyramidal and showed 2-3 long (50-100 μm) immunoreactive dendrites. Any immunoreactive axons containing pantothenic acid were detected. The very restricted anatomical distribution of the pantothenic acid suggests that this vitamin could be involved in some specific neurophysiological mechanisms.

NO-tryptophan: a new small molecule located in the rat brain.

A highly specific monoclonal antibody directed against nitric oxide-tryptophan (NO-W) with good affinity (10-9 M) and specificity was developed. In the rat brain, using an indirect immunoperoxidase technique, cell bodies containing NO-W were exclusively found in the intermediate and dorsal parts of the lateral septal nucleus. No immunoreactive fibres were found in the rat brain. This work reports the first visualization and the morphological characteristics of cell bodies containing NO-W in the mammalian brain. The restricted distribution of NO-W in the rat brain suggests that this molecule could be involved in specific physiological mechanisms.

Distribution of Neurotensin and Somatostatin-28 (1-12) in the Minipig Brainstem.

Using an indirect immunoperoxidase technique, an in depth study has been carried out for the first time on the distribution of fibres and cell bodies containing neurotensin and somatostatin-28 (1-12) (SOM) in the minipig brainstem. The animals used were not treated with colchicine. The distribution of neurotensin- and SOM-immunoreactive fibres was seen to be quite similar and was moderate in the minipig brainstem: a close anatomical relationship between both neuropeptides was observed. The distribution of cell bodies containing neurotensin or SOM was quite different and restricted. Cell bodies containing neurotensin were found in four brainstem nuclei: nucleus centralis raphae, nucleus dorsalis raphae, in the pars centralis of the nucleus tractus spinalis nervi trigemini and in the nucleus ventralis raphae. Cell bodies containing SOM were found in six nuclei/regions of the brainstem: nucleus ambiguus, nucleus dorsalis motorius nervi vagus, formatio reticularis, nucleus parabrachialis medialis, nucleus reticularis lateralis and nucleus ventralis raphae. According to the observed anatomical distribution of the immunoreactive structures containing neurotensin or SOM, the peptides could be involved in sleep-waking, nociceptive, gustatory, motor, respiratory and autonomic mechanisms.

Mapping of somatostatin-28 (1-12) in the alpaca (Lama pacos) brainstem.

Using an indirect immunoperoxidase technique, we studied the distribution of cell bodies and fibers containing somatostatin-28 (1-12) in the alpaca brainstem. Immunoreactive fibers were widely distributed throughout the whole brainstem: 34 brainstem nuclei/regions showed a high or a moderate density of these fibers. Perikarya containing the peptide were widely distributed throughout the mesencephalon, pons and medulla oblongata. Cell bodies containing somatostatin-28 (1-12) were observed in the lateral and medial divisions of the marginal nucleus of the brachium conjunctivum, reticular formation (mesencephalon, pons and medulla oblongata), inferior colliculus, periaqueductal gray, superior colliculus, pericentral division of the dorsal tegmental nucleus, interpeduncular nucleus, nucleus of the trapezoid body, vestibular nucleus, motor dorsal nucleus of the vagus, nucleus of the solitary tract, nucleus praepositus hypoglossi, and in the substantia nigra. This widespread distribution indicates that somatostatin-28 (1-12) is involved in multiple physiological actions in the alpaca brainstem.

Differential Cytokine Expression and Regulatory Cells in Patients with Primary and Secondary Sjögren's Syndrome

Sjögren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration of the salivary and lacrimal glands. The aim of the study was to characterize and compare the presence of diverse cytokines and regulatory T and B cells in lip minor salivary gland (MSG) biopsies from patients with primary Sjögren's syndrome (pSS), secondary SS (sSS), and patients with connective tissue disease (CTD) without (w/o) SS. We included samples of MSG from 15 pSS, 24 sSS (six scleroderma, nine rheumatoid arthritis and nine lupus patients) and 15 patients with CTD w/o SS. Tissues were examined by an indirect immunoperoxidase technique (goat polyclonal anti-human IL-19, goat polyclonal anti-human IL-22 or mouse monoclonal anti-human IL-24). To determine the subpopulation of CD4(+)/IL-17A(+)-, CD4(+)/IL-4(+)-, CD4(+)/IFN-ɣ(+)-expressing T cells, CD25(+)/Foxp3(+) Treg cells and CD20(+)/IL-10(+)-producing B cell subset, a double-staining procedure was performed. We estimated the mean percentage of positively staining cells in two fields per sample. CD4(+)/IFN-ɣ(+), CD4(+)/IL-4(+) and IL-22(+) cell percentages were elevated in both SS varieties; however, the cells were more prevalent in pSS. Patients with pSS had a high number of CD4(+)/IL-17A(+) and IL-19(+) T cells and a lower percentage of IL-24(+) cells (P < 0.05). The Treg and IL-10-producing B cells were increased in pSS (P < 0.05). Concluding, in our patients, a pro-inflammatory and regulatory balance coexists in SS, being both responses more intense in pSS. The explanation of these differences may be related to disease activity, disease duration and treatment.

Immunohistochemical mapping of pro-opiomelanocortin- and pro-dynorphin-derived peptides in the alpaca (Lamapacos) diencephalon

Using an indirect immunoperoxidase technique, we studied the distribution of cell bodies and fibres containing non-opioid peptides (adrenocorticotropin hormone (ACTH), alpha-melanocyte-stimulating hormone) and opioid peptides (beta-endorphin (1–27), alpha-neo-endorphin, leucine-enkephalin) in the alpaca diencephalon. No immunoreactive cell bodies containing ACTH were found. Perikarya containing the other four peptides were observed exclusively in the hypothalamus and their distribution was restricted. Perikarya containing alpha-melanocyte-stimulating hormone or alpha-neo-endorphin showed a more widespread distribution than those containing leucine-enkephalin or beta-endorphin (1–27). Cell bodies containing pro-opiomelanocortin-derived peptides were observed in the arcuate nucleus, anterior and lateral hypothalamic areas and in the ventromedial and supraoptic hypothalamic nuclei, whereas perikarya containing alpha-neo-endorphin (a pro-dynorphin-derived peptide) were found in the arcuate nucleus, dorsal and lateral hypothalamic areas, and in the paraventricular, ventromedial and supraoptic hypothalamic nuclei. Immunoreactive cell bodies containing leucine-enkephalin were found in the lateral hypothalamic area and in the paraventricular hypothalamic nucleus. Immunoreactive fibres expressing pro-opiomelanocortin-derived peptides were more numerous than those expressing pro-dynorphin-derived peptides. A close anatomical relationship was observed: in all the diencephalic nuclei in which beta-endorphin (1–27)-immunoreactive fibres were found, fibres containing alpha-melanocyte-stimulating hormone or alpha-neo-endorphin were also observed. Fibres containing beta-endorphin (1–27), alpha-melanocyte-stimulating hormone or alpha-neo-endorphin were widely distributed throughout the diencephalon, but fibres containing ACTH or leucine-enkephalin showed a moderate distribution. The distribution of the five peptides studied here is also compared with that reported previously in other mammalian species. The widespread distribution observed indicates that both the pro-dynorphin and the pro-opiomelanocortin systems are involved in multiple physiological actions (e.g., food intake, thermoregulation, neuroendocrine and reproductive mechanisms) in the alpaca diencephalon.

Distribution of CGRP in the minipig brainstem

For the first time, an in-depth study has been made of the distribution of fibers and cell bodies containing calcitonin gene-related peptide (CGRP) in the minipig brainstem using an indirect immunoperoxidase technique. The animals studied were not treated with colchicine. Cell bodies containing CGRP were found in 20 nuclei/regions of the brainstem. These perikarya were located in somatomotor, brachiomotor and raphae nuclei, nucleus ambiguus, substantia nigra, nucleus reticularis tegmenti pontis, nucleus prepositus hypoglossi, nuclei olivaris inferior and superior, nuclei pontis, formatio reticularis, nucleus dorsalis tegmenti of Gudden, and in the nucleus reticularis lateralis. Fourteen of the 20 brainstem nuclei showed a high density of immunoreactive cell bodies. In comparison with other species, the minipig, together with the rat, show the most widespread distribution of cell bodies containing CGRP in the mammalian brainstem. Immunoreactive fibers were also observed in the brainstem. However, in the minipig brainstem the density of these fibers is low, as in many brainstem nuclei only single immunoreactive fibers were observed. A high density of immunoreactive fibers was only observed in the pars caudalis of the nucleus tractus spinalis nervi trigemini and in the nucleus ventralis tegmenti of Gudden. According to the observed anatomical distribution of the immunoreactive structures containing CGRP, the peptide could be involved in motor, somatosensory, gustative, and autonomic mechanisms.

Mapping of Neurotensin in the Alpaca (Lama pacos) Brainstem

We studied the distribution of cell bodies and fibres containing neurotensin (NT) in the brainstem of the alpaca using an indirect immunoperoxidase technique. Immunoreactive fibres were widely distributed throughout the brainstem, whereas the distribution of cell bodies was less widespread. Immunoreactive perikarya were only found in the mesencephalic and bulbar reticular formation, periaqueductal grey, nucleus of the solitary tract, laminar spinal trigeminal nucleus and in the inferior colliculus. A high density of fibres containing NT was found in the dorsal nucleus of the raphe, marginal nucleus of the brachium conjunctivum, locus coeruleus, inferior colliculus, inter-peduncular nucleus, substantia nigra, periaqueductal grey, reticular formation of the mesencephalon, pons and medulla oblongata, nucleus of the solitary tract, laminar spinal trigeminal nucleus, hypoglossal nucleus, inferior central nucleus and in the tegmental reticular nucleus. The widespread distribution indicates that NT might be involved in multiple physiological actions in the alpaca brainstem; this must be investigated in the future as alpacas lives from 0 m above sea level to altitudes of up 5000 m and hence the involvement of this neuropeptide in special and unique regulatory physiological mechanisms could be suggested.