Mass Spectrometry for Identification, Monitoring, and Minimal Residual Disease Detection of M-Proteins.

BACKGROUND: Monoclonal gammopathies (MGs) are plasma cell disorders defined by the clonal expansion of plasma cells, resulting in the characteristic excretion of a monoclonal immunoglobulin (M-protein). M-protein detection and quantification are integral parts of the diagnosis and monitoring of MGs. Novel treatment modalities impose new challenges on the traditional electrophoretic and immunochemical methods that are routinely used for M-protein diagnostics, such as interferences from therapeutic monoclonal antibodies and the need for increased analytical sensitivity to measure minimal residual disease. CONTENT: Mass spectrometry (MS) is ideally suited to accurate mass measurements or targeted measurement of unique clonotypic peptide fragments. Based on these features, MS-based methods allow for the analytically sensitive measurement of the patient-specific M-protein. SUMMARY: This review provides a comprehensive overview of the MS methods that have been developed recently to detect, characterize, and quantify M-proteins. The advantages and disadvantages of using these techniques in clinical practice and the impact they will have on the management of patients with MGs are discussed.

Measuring methotrexate polyglutamates in red blood cells: a new LC-MS/MS-based method.

The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTXPGn). We developed a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate MTXPGn in red blood cells using stable-isotope-labelled internal standards. Samples were analyzed by LC-ESI-MS/MS using a Waters Acquity UPLC BEH C18 column with a 5-100% organic gradient of 10 mM ammonium bicarbonate (pH 10) and methanol. The analysis consisted of simple sample preparation and a 6-min run time. Detection was done using a Waters Acquity UPLC coupled to a Waters Quattro Premier XE with electrospray ionization operating in the positive ionization mode. Assay validation was performed following recent Food and Drug Administration guidelines. The method was linear from 1-1,000 nM for all MTXPGn (R(2) > 0.99). The coefficient of variation ranged from 1-4% for intraday precision and 6-15% for interday precision. Samples were stable for at least 1 month at -80 °C. Recovery ranged from 98-100%, and the relative matrix-effect varied from 95-99%. The lower limit of quantitation was 1 nM for each MTXPGn. Fifty patient samples from the tREACH study were analyzed. The MTXPGn concentration and distribution of these samples were comparable with values reported in literature. The developed LC-ESI-MS/MS method for the quantitative measurement of MTXPGn in red blood cells is both sensitive and precise within the clinically relevant range. The method can be easily applied in clinical laboratories due to the combination of simple pre-treatment with robust LC-ESI-MS/MS.

Dataset from a proteomics analysis of tumor antigens shared between an allogenic tumor cell lysate vaccine and pancreatic tumor tissue.

The data described was acquired as part of a clinical study with the aim to investigate the potential of tumor-reactive T-cell response as response to vaccination of pancreatic cancer patients with an allogenic tumor cell lysate vaccine (Lau et al., 2022). Proteomics analysis was carried out to identify tumor antigens that are shared between the allogeneic tumor cell lysate used for the vaccine and pancreatic ductal adenocarcinoma (PDAC) tissue samples. To this objective, cell lysates of the vaccine and of nine tissue samples were enzymatically digested and isotopically labeled with tandem mass tags (TMT) in a so-called six-plex manner (Thermo Fisher Scientific). Three pools were prepared by mixing the samples according to their TMT-labels. Subsequently, the three sample pools were fractionated into 24 fractions with high-pH reversed phase chromatography. These fractions were first analyzed on a nano-liquid chromatography (LC) system online coupled to a high-resolution Eclipse Orbitrap mass spectrometer (MS) equipped with a high-field asymmetric-waveform ion-mobility spectrometry (FAIMS) source using a data-dependent MS2 shotgun method. Overall, 126,618 unique peptide sequences, on basis of 768,638 peptide spectra matches and corresponding to 7,597 protein groups, were identified in the total sample set including 61 tumor antigens (Supplement Table S2 in Lau et al. 2022) that were prioritized by Cheever and co-workers as vaccine target antigens on basis of a series of objective criteria (Cheever et al., 2009). In the second phase of the experiment, this set of tumor antigens was targeted using a serial precursor selection (SPS) MS3 method. From this data, ion trap MS2 and Orbitrap MS3 fragment spectra were extracted for peptide identification (protein sequence database-dependent search) and relative quantification using the TMT labels, respectively. The dataset ultimately allowed the identification and quantification of 51 proteins and 163 related peptide precursors with the TMT labels (see Fig. 2B and Supplemental Fig. 8, Lau et al. 2022).

Autologous dendritic cells pulsed with allogeneic tumour cell lysate induce tumour-reactive T-cell responses in patients with pancreatic cancer: A phase I study.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is notorious for its poor prognosis even after curative resection. Responses to immunotherapy are rare and related to inadequate T-cell priming. We previously demonstrated the potency of allogeneic lysate-dendritic cell (DC) vaccination in a preclinical model. Here we translate this concept to patients. METHODS: In this phase I study, patients with resected PDAC were included when they demonstrated no radiologic signs of recurrence after standard-of-care treatment. Allogeneic tumour lysate-loaded autologous monocyte-derived DCs were injected at weeks 0, 2, 4 and at months 3 and 6. Objectives are feasibility, safety and immunogenicity of allogeneic tumour-DCs. The presence of tumour antigens shared between the vaccine and patient tumours was investigated. Immunological analyses were performed on peripheral blood, skin and tumour. RESULTS: Ten patients were included. DC production and administration were successful. All patients experienced a grade 1 injection-site and infusion-related reaction. Two patients experienced a grade 2 fever and 1 patient experienced a grade 3 dyspnoea. No vaccine-related serious adverse events were observed. Shared tumour antigens were found between the vaccine and patient tumours. All evaluated patients displayed a vaccine-induced response indicated by increased frequencies of Ki67+ and activated PD-1+ circulating T-cells. In addition, treatment-induced T-cell reactivity to autologous tumour of study patients was detected. Seven out of ten patients have not experienced disease recurrence or progression at a median follow-up of 25 months (15-32 months). CONCLUSION: Allogeneic tumour lysate-DC treatment is feasible, safe and induces immune reactivity to PDAC expressed antigens.

Comparative proteomic analysis of human mesenchymal stromal cell behavior on calcium phosphate ceramics with different osteoinductive potential.

In recent years, synthetic calcium phosphate (CaP) ceramics have emerged as an alternative to bone grafts in the treatment of large critical-sized bone defects. To successfully substitute for bone grafts, materials must be osteoinductive, that is, they must induce osteogenic differentiation and subsequent bone formation in vivo. Although a set of osteoinductive CaP ceramics has been developed, the precise biological mechanism by which a material directs cells toward osteogenesis and the role of individual chemical and physical properties in this mechanism remain incompletely understood. Here, we used proteomics to compare serum protein adsorption to two CaP ceramics with different osteoinductive potential, namely an osteoinductive ß-tricalcium phosphate (TCP) and a non-osteoinductive hydroxyapatite (HA). Moreover, we analyzed the protein profiles of human mesenchymal stromal cells (hMSCs) cultured on these two ceramics. The serum protein adsorption experiments in the absence of cells highlighted the proteins that are highly abundant in the serum and/or have a high affinity to CaP. The extent of adsorption was suggested to be affected by the available surface area for binding and by the ion exchange dynamics on the surface. Several proteins were uniquely expressed by hMSCs on TCP and HA surfaces. Proteins identified as enriched on TCP were involved in processes related to wound healing, cell proliferation, and the production of extracellular matrix. On the other hand, proteins that were enriched on HA were involved in processes related to protein production, translation, localization, and secretion. In addition, we performed a separate proteomics analysis on TCP, HA, and two biphasic calcium phosphates with known osteoinductive potential and performed a clustering analysis on a combination of a set of proteins found to be enriched on osteoinductive materials with a set of proteins already known to be involved in osteogenesis. This yielded two protein networks potentially involved in the process of osteoinduction - one consisting of collagen fragments and collagen-related enzymes and a second consisting of endopeptidase inhibitors and regulatory proteins. The results of this study show that protein profiling can be a useful tool to help understand the effect of biomaterial properties on the interactions between a biomaterial and a biological system. Such understanding will contribute to the design and development of improved biomaterials for (bone) regenerative therapies.

Serum proteomic patterns for ovarian cancer monitoring.

We set out to discover ovarian cancer biomarkers useful for monitoring progression during and after chemotherapy and possibly for diagnosis. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to create serum protein profiles of ovarian cancer patients before chemotherapy or at progression (n = 51) (trial initiated by the Gynecological Cancer Cooperative Group of the European Organization for Research and Treatment of Cancer trial) that were compared with those of healthy individuals (n = 31). In addition, sera profiles from ovarian cancer patients after chemotherapy (n = 12) were compared with those of ovarian cancer patients at progression (n = 24). One of the discovered biomarkers was identified and subsequently confirmed and validated using enzyme-linked immunosorbent assay (ELISA). Eight primary (sens = 94%, spec = 97%, P < 0.0001) and seven progression tumor biomarkers (sens = 91%, spec = 97%, P < 0.0001) were discovered. In addition, we discovered eight potential progression monitoring biomarkers (sens = 75%, spec = 83%, P = 0.0008) of which one, a biomarker of 11.7 kd, was further identified as serum amyloid A1. Independent validation (ELISA) showed an elevated expression of this protein at relapse in four of the seven ovarian cancer patients tested. Combining the eight newly discovered progression monitoring biomarkers with CA125 resulted in a clear increase of the sensitivity (91-100%). These biomarkers, in combination with for instance CA125, should be validated in large ovarian cancer and control groups. The resulting multimarker assay could be suitable for disease monitoring during and after therapy and might also be useful for ovarian cancer screening.

Relationship between natural killer cell susceptibility and metastasis of human uveal melanoma cells in a murine model.

PURPOSE: The purpose of this study was to determine the susceptibility of human uveal melanoma cells to in vitro and in vivo natural killer (NK) cell-mediated cytolysis and to determine if NK cells influence metastasis from the eye. METHODS: Four human uveal melanoma cell lines and one melanoma cell line derived from a metastatic lesion from a patient with uveal melanoma were tested for in vitro and in vivo NK cell-mediated lysis in a mouse model. Major histocompatibility complex (MHC) class I antigen expression was evaluated by flow cytometry. The role of NK cells in controlling the metastasis of uveal melanoma cells from the eye to the liver was examined in nude mice. RESULTS: Sensitivity to in vitro and in vivo lysis by human and murine NK cells was correlated with reduced expression of MHC class I antigens. Uveal melanoma lines expressing normal MHC class I antigen expression were insensitive to NK cell-mediated lysis, both in vitro and in vivo. Metastasis of uveal melanoma cells was inhibited by NK cell activity because disruption of in vivo NK function produced a sharp increase in the spontaneous metastasis of intraocular melanomas in nude mice. CONCLUSIONS: There is considerable variation in the susceptibility of human uveal melanomas to NK cell-mediated cytolysis. Susceptibility is closely correlated with reduced expression of MHC class I antigen expression. Disruption of NK cell function significantly increases the development of hepatic metastases from human uveal melanoma cells.

Analysis of carbohydrate structures in basal laminar deposit in aging human maculae.

PURPOSE: To analyze carbohydrate structures in basal laminar deposit (BLD), an extracellular material that accumulates between the retinal pigment epithelium (RPE) and Bruch's membrane. BLD has been shown to correlate positively with visual loss in age-related macular degeneration. METHODS: Thirteen postmortem human maculae with BLD were histochemically examined by light microscopy using the monoclonal antibody HNK-1 and seven lectins; canavalia ensiformis (ConA), soybean agglutinin (SBA), wheat germ agglutinin (WGA), dolichos bifloris (DBA), ulex europaeus (UEA-I), ricinius communis agglutinin I (RCA-I), and peanut agglutinin (PNA). Three maculae were stained with polyclonal antibodies against laminin and collagen type IV. RESULTS: BLD was exclusively stained by DBA and SBA, whereas Con A, WGA, UEA-I, RCA-I, and HNK-1 stained various other structures in the human macula as well. The main part of the BLD adjacent to Bruch's membrane stained with these lectins and the monoclonal antibody HNK-1, whereas only a small part of the BLD adjoining the RPE stained with antibodies against laminin and collagen type IV. Drusen stained neither with any lectin nor with any antibody. CONCLUSIONS: DBA and SBA, which bind specifically to an alpha-D-GalNAc moiety, are specific markers for the light-microscopic detection of BLD in human macular tissue. Furthermore, the authors conclude that BLD contains several carbohydrate structures other than the carbohydrate moieties on laminin and collagen type IV. If drusen contain carbohydrate structures, these must be different from those in BLD.

Association between NM23-H1 gene expression and metastasis of human uveal melanoma in an animal model.

PURPOSE: To evaluate the role of nm23 gene expression in the development of metastases of human uveal melanomas in an animal model. METHODS: Seven human uveal melanoma cell lines and two murine skin melanoma cell lines were subjected to Northern blot analysis for the detection of nm23-H1 mRNA and to immuno-histochemistry to detect nm23 antigen. Each tumor cell line was transplanted intracamerally into nude mice, and the metastatic behavior was evaluated by histopathologic analysis of the livers and by determining host survival times. RESULTS: There was a strong inverse correlation between the levels of nm23 mRNA expression and nm23 antigen expression and the development of metastases of all seven human uveal melanomas and both murine skin melanomas transplanted intracamerally. Host survival time also was correlated with the degree of nm23 gene expression. CONCLUSIONS: The expression of nm23 mRNA and nm23 antigen in human uveal melanomas is correlated closely with reduced metastatic behavior in experimental animals and may serve as a sensitive prognostic indicator of malignancy and survival in patients with uveal melanomas.

Neural cell adhesion molecule distribution in primary and metastatic uveal melanoma.

Tumor cell adhesion, detachment, and aggregation play an important part in tumor invasion and metastasis, and a variety of cell adhesion molecules have been found on tumor cells. Cell adhesion molecules, including those of the immunoglobulin superfamily, are associated with the development of metastatic behavior in cutaneous melanomas. The neural cell adhesion molecule (NCAM) belongs to this family. To investigate its possible role in the development metastatic behavior of uveal melanomas, the authors studied immunohistochemically the expression of NCAM by using an antibody that recognizes all three major isoforms of NCAM and an antibody that recognizes the HNK-1 epitope present on some isoforms of NCAM. The authors studied 32 primary uveal melanomas from 32 patients (among these, 12 were rapidly metastasizing and 16 slowly metastasizing) and 29 metastases from 19 patients. From 13 patients the primary, as well as the metastatic, tumors were available. With one exception, all HNK-1 positive primary and metastatic tumors were also positive for NCAM. NCAM was significantly more expressed in aggressive, rapidly metastasizing primary tumors (P = .02 and .04, respectively) and in metastases. HNK-1 was significantly (P = .04) more expressed in larger tumors. In liver metastases HNK-1 immunoreactivity was significantly (P = .005) less frequently expressed than NCAM. Therefore, NCAM isoforms that lack the HNK-1 epitope might play a role in the organ specific metastatic behavior of uveal melanomas.

Identification of glycosaminoglycans in age-related macular deposits.

We investigated the presence and localization of glycosaminoglycans in basal laminar deposit and drusen in age-related maculopathy. Conventional histological staining techniques and monoclonal antibodies specific for several glycosaminoglycans were used on paraffin-embedded human maculae. Furthermore, macular homogenates were analyzed with two-dimensional electrophoresis. Quantitative analysis of glycosaminoglycans was done spectrophotometrically using dimethylmethylene blue. Immunohistochemically, all basal laminar deposit stained positive for chondroitin 4-sulfate and focally positive for heparan sulfate proteoglycan. Drusen were not stained with any of the monoclonal antibodies. With two-dimensional electrophoresis, it was demonstrated that macular extracts with and without age-related maculopathy contained chondroitin sulfate. Heparan sulfate was only expressed in maculae with age-related maculopathy. The total amount of glycosaminoglycans was significantly higher in maculae with basal laminar deposit than in maculae without basal laminar deposit (P = .001). There were significant differences in the amount and composition of glycosaminoglycans between maculae with and without age-related maculopathy.

No demonstrated effect of pre-enucleation irradiation on survival of patients with uveal melanoma.

PURPOSE: To evaluate the hypothetical effect of pre-enucleation irradiation on survival of patients with uveal melanoma. METHODS: In a prospective study between 1978 and 1990, 145 patients with uveal melanoma were treated by irradiation in two fractions of 4 Gy before enucleation. A historical control group of 89 patients with uveal melanoma treated by enucleation alone was operated on between 1971 and 1990. Patients were followed up until December 1992 or until death. The mean follow-up period was 65 months in the irradiated group and 88 months in the control group. RESULTS: The preoperatively irradiated group of patients showed no significant improvement of the survival rate after 7 1/2 years (75.9%) compared with the control group (72.1%). Preoperative irradiation was not associated with survival (P = .93), as assessed by Cox proportional hazard analysis, adjusted for age, gender, tumor location, tumor size, cell type, and year of enucleation. Women in both the irradiated and control groups had a better prognosis than men (P = .002). CONCLUSION: Preoperative irradiation in this nonrandomized study had no effect on survival of patients with uveal melanoma.

HNK-1 antigens on uveal and cutaneous melanoma cell lines.

The HNK-1 epitope has been associated with the metastatic behaviour of uveal melanomas. We characterized HNK-1 antigens on four human uveal (primary and metastatic) and two primary cutaneous melanoma cell lines by immunocytochemistry and Western blot analysis. We also determined the involvement of the HNK-1 epitope in cell-cell interactions on a matrigel layer. Three uveal melanoma cell lines (one primary and two metastatic) and one cutaneous melanoma cell line showed HNK-1 expression by immunocytochemistry. On matrigel, only the HNK-1-positive cutaneous melanoma cell line Bowes grew in a honeycomb-like structure which disappeared after adding HNK-1 antibodies to the culture medium. Immunoblot analysis of the primary uveal melanoma cell line EOM-3 revealed five HNK-1-positive protein bands with apparent molecular weights of 200, 160, 115, 95 and 75 kDa. The cutaneous melanoma cell line Bowes showed three HNK-1-positive protein bands with apparent molecular weights of 150, 135 and 90 kDa. This study shows that two uveal (primary and metastatic) and one primary cutaneous melanoma cell lines express HNK-1 antigens on immunoblot. Only in the HNK-1-positive cutaneous melanoma cell line Bowes did the HNK-1 epitope have a function in intercellular adhesion. Although the primary uveal melanoma cell line EOM-3 showed a similar HNK-1 immunoreactivity, we could not demonstrate HNK-1-mediated cell adhesion. On immunoblot, the two cell lines displayed different HNK-1 antigens, which may explain the difference in cell adhesion.

Expression of MAGE, gp100 and tyrosinase genes in uveal melanoma cell lines.

In order to determine the possible use of uveal melanoma cell lines as stimulators in immunotherapy, we evaluated the expression of the human genes for MAGE-1, -2 and -3, gp100 and tyrosinase in uveal melanoma cell lines. mRNA expression of the MAGE-1, -2 and -3, gp100 and tyrosinase genes and the HLA class I specificity were determined in five primary and three metastatic uveal melanoma cell lines. Expression of the examined genes was heterogeneous in the primary and metastatic cell lines. The cell lines OCM-1 and OMM-1 expressed MAGE-1, -2 and -3, whereas EOM-3, MEL202, 92-1 and OMM-3 were negative for these antigens. gp100 was expressed in all cell lines, and tyrosinase in all but three (EOM-29, OMM-2 and OMM-3). Except for EOM-3, the HLA-A type of all the cell lines could be determined by complement-dependent microlymphocytotoxicity assay. Since at least two melanoma-associated antigens can be found in uveal melanoma cell lines, as well as the HLA class I molecules, these cell lines may be applicable as immunogens for specific immunotherapy against metastatic uveal melanoma.

The distribution and characterization of HNK-1 antigens in the developing avian heart.

The heart originates from splanchnic mesoderm and to a lesser extent from neural crest cells. The HNK-1 monoclonal antibody is a marker for early migrating neural crest cells, but reacts also with structures which are not derived from the neural crest. We investigated whether heart structures are HNK-1 positive before neural crest cells colonize these target tissues. To that end, we determined the HNK-1 antigen expression in the developing avian heart on immunohistochemical sections and on Western blots. The HNK-1 immunoreactivity in the developing chick heart is compared with data from literature on the localization of neural crest cells in chick/quail chimeras. Structures with neural crest contribution, including parts of the early outflow tract and the related endocardial cushions, the primordia of the semilunar valve leaflets and the aorticopulmonary septum were HNK-1 positive. Furthermore, other structures were HNK-1 positive, such as the atrioventricular cushions, the wall of the sinus venosus at stage HH 15 through 21, parts of the endocardium at E3, parts of the myocardium at E6, and the extracellular matrix in the myocardial base of the semilunar valves at E14. HNK-1 expression was particularly observed in morphologically dynamic regions such as the developing valves, the outflow tract cushion, the developing conduction system and the autonomic nervous system of the heart. We observed that atrioventricular endocardial cushions are HNK-1 positive. We conclude that: a HNK-1 immunoreactivity does not always coincide with the presence of neural crest cells or their derivatives; (2) the outflow tract cushions and atrioventricular endocardial cushions are HNK-1 positive before neural crest cells are expected (stage HH 19) to enter the endocardial cushions of the outflow tract; (3) the observed spatio-temporal HNK-1 patterns observed in the developing heart correspond with various HNK-1 antigens. Apart from a constant pattern of HNK-1 antigens during development, stage-dependent HNK-1 antigens were also found.

Colonization characteristics of enteric neural crest cells: embryological aspects of Hirschsprung's disease.

This study explores the development of the enteric nervous system in avian embryos. Particular emphasis was given to colonization characteristics of neural crest cells present in primitive enteric ganglia. By coculturing neuronal and aneuronal gut of quail and chicken embryos, we investigated if and when neural crest cells in primitive enteric ganglia could detach from these ganglia, migrate, and colonize adjacent chicken gut. Quail neural crest cells were identified using the quail nucleolar marker and the HNK-1 antibody. Enteric neurons were identified using three monoclonal antibodies directed against neurofilament proteins. We found that neural crest cells detached from primitive ganglia in neuronal quail gut from E6 till E9, whereas neural crest cells did not leave enteric ganglia from E10 gut. These observations show that there is a transient phase during which enteric neural crest cells can leave the gut. To determine whether neural crest cells could colonize neuronal gut we cocultured neuronal gut or the neural primordium and neuronal chicken gut (E11). We found that quail neural crest cells do not colonize neuronal E11 gut, whereas they do colonize aneuronal gut of the same age. We suggest that aneuronal gut attracts neural crest cells by diffusing factors.

Differences in phosphorylation state of neurofilament proteins in ganglionic and aganglionic bowel segments of children with Hirschsprung's disease.

Hirschsprung's disease is characterized by the absence of enteric neurons in the myenteric and submucosal plexus and the presence of many unmyelinated axons, visible in ganglion like structures, in the aganglionic part of the bowel. In previous studies we showed that the immunoreactivity of a monoclonal antibody (2F11) specific for neurofilament proteins is increased in aganglionic bowel segments. We now investigated whether the increased neurofilament protein staining results from an increase in neurofilament protein immunoreactivity in the aganglionic segment or if it is also related to differences in the phosphorylation state of neurofilament proteins. Bowel resection specimens of patients with Hirschsprung's disease and control patients were investigated by immunohistochemical techniques using a panel of different monoclonal antibodies that are specific for neurofilament proteins and have well known reaction patterns against different phosphorylated epitopes present on two neurofilament proteins, the middle (NF-M) and the high (NF-H) molecular weight subunit. For comparison the specimens were also stained for acetylcholinesterase, neuron-specific enolase (NSE), S-100, and glial fibrillary acidic protein (GFAP). Immunostaining with this panel of antineurofilament-antibodies showed differences in the phosphorylation state of neurofilament proteins in the aganglionic and the ganglionic bowel segments of patients with Hirschsprung's disease. These changes involved the phosphorylation state of these proteins and the ratio of NF-H and NF-M in neurofilament proteins. Staining with NSE and S-100 showed no significant differences between Hirschsprung's disease patients and control patients. We surmise that during the ingrowth and differentiation of hypertrophic axons the composition of neurofilament proteins formed in the aganglionic bowel segment differs from the neurofilament proteins formed in the ganglionic and control bowel segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation and malformation of the enteric nervous system in mice: an organ culture study.

Despite some progress in the treatment of congenital malformations of the enteric nervous system, there is no knowledge about the pathogenesis. The study of the normal formation of the enteric nervous system is hampered by the difficulty of manipulating and culturing mammalian embryos and their organs. Three methods to culture bowel explants of murine embryos, (chorioallantoic membrane grafting, organotypic tissue culture, and renal subcapsular space grafting) were compared. The three-dimensional cytoarchitecture of the bowel developed almost normally in the renal subcapsular space cultures. Using this culture system, it was found that neural crest cells colonize the murine bowel in distinct phases. The distal bowel was colonized at the 13th day of development. In a spontaneous mouse mutant model for intestinal aganglionosis, the lethal spotted mouse, the colonization of the distal 2 mm of the bowel did not occur at E13.

The Han:SPRD rat is not a genetic model of human autosomal dominant polycystic kidney disease type 1.

Human autosomal dominant polycystic kidney disease (ADPKD) is a high incidence disorder leading to renal failure in many patients. The majority of cases results from a mutation in the PKD1 gene. The only well documented animal model of ADPKD is the Han:SPRD-Pkd strain. Its genetic basis is unknown as yet. In the current study we determined whether the disease in these rats is genetically linked to the rat homologue of the PKD1 gene. We used the protamine gene as a polymorphic marker (Prm1) of the PKD1 region. Matings of Han:SPRD-Pkd with BB rats and backcross of the offspring with BB yielded animals informative for linkage analysis. This analysis revealed random segregation of the defect and the Prm1 marker, indicating that the model is not caused by a mutation in the PKD1 gene. We conclude that the Han:SPRD-Pkd rat strain is not a genetic model of PKD1.

Trophoblast calcyclin is elevated in placental tissue from patients with early pre-eclampsia.

The aetiology of pre-eclampsia is thought to originate from aberrant spiral artery remodelling and invasion evoking cellular oxidative stress. Previously, we discovered differentially expressed proteins in trophoblast cells of pre-eclamptic pregnancies. One of these proteins is calcyclin (S100A6); a Ca(2+)-binding protein associated with cellular stress response. By immunohistochemistry on formalin-fixed paraffin-embedded placental tissue, calcyclin expression was compared between women with early pre-eclampsia (n=72) and non-hypertensive control patients (n=66) (χ(2), p=0.006) blindly by two observers. Significantly more intense staining was seen in trophoblast cells of pre-eclamptic pregnancies compared to control placentas suggesting that trophoblast calcyclin is elevated in early pregnancy.