High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses.

BACKGROUND: The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. RESULTS: Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. CONCLUSIONS: In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.

Quantitative analysis of histidine decarboxylase gene (hdcA) transcription and histamine production by Streptococcus thermophilus PRI60 under conditions relevant to cheese making.

This study evaluated the influence of parameters relevant for cheese making on histamine formation by Streptococcus thermophilus. Strains possessing a histidine decarboxylase (hdcA) gene represented 6% of the dairy isolates screened. The most histaminogenic, S. thermophilus PRI60, exhibited in skim milk a high basal level of expression of hdcA, upregulation in the presence of free histidine and salt, and repression after thermization. HdcA activity persisted in cell extracts, indicating that histamine might accumulate after cell lysis in cheese.

A quantitative in situ hybridization protocol for formalin-fixed paraffin-embedded archival post-mortem human brain tissue.

The use of radioactive in situ hybridization (ISH) to quantitatively determine low-to-moderate abundant mRNA expression in formalin-fixed, paraffin-embedded archival post-mortem human brain tissue is often limited by non-specific-deposits, visible as speckles. In the present study, optimal hybridization conditions were achieved for quantifying the mRNA expression of histidine decarboxylase (HDC) by a number of alterations in a routine protocol, which included (1) during purification of the oligo-probes, glycogen was omitted as a carrier for precipitation, (2) after precipitation, the labeled probe contained within the pellet was first dissolved in water instead of in hybridization buffer (HBF), (3) during hybridization, the dithiothreitol (DTT) concentration was increased from 200 to 800 mM in HBF, and (4) stringencies during hybridization and post-hybridization washes were increased by increasing the temperature. The effect of the adjustment was quantified on adjacent sections from 18 subjects (9 with Parkinson's disease and 9 controls), by comparing the data from the standard and new protocol. The results showed that the improved protocol brought about significantly clearer background with higher signal-to-noise ratios (p=0.001). We propose that this protocol is also applicable for detection of other lower-abundant genes in human brain tissue and probably in other tissues as well. In the present study, this is not only illustrated for HDC ISH, but also for corticotrophin-releasing hormone mRNA expression in the hypothalamic paraventricular nucleus.

Histamine and histidine decarboxylase in the olfactory system and brain of the common cuttlefish Sepia officinalis (Linnaeus, 1758).

Cephalopods are radically different from any other invertebrate. Their molluscan heritage, innovative nervous system, and specialized behaviors create a unique blend of characteristics that are sometimes reminiscent of vertebrate features. For example, despite differences in the organization and development of their nervous systems, both vertebrates and cephalopods use many of the same neurotransmitters. One neurotransmitter, histamine (HA), has been well studied in both vertebrates and invertebrates, including molluscs. While HA was previously suggested to be present in the cephalopod central nervous system (CNS), Scaros, Croll, and Baratte only recently described the localization of HA in the olfactory system of the cuttlefish Sepia officinalis. Here, we describe the location of HA using an anti-HA antibody and a probe for histidine decarboxylase (HDC), a synthetic enzyme for HA. We extended previous descriptions of HA in the olfactory organ, nerve, and lobe, and describe HDC staining in the same regions. We found HDC-positive cell populations throughout the CNS, including the optic gland and the peduncle, optic, dorso-lateral, basal, subvertical, frontal, magnocellular, and buccal lobes. The distribution of HA in the olfactory system of S. officinalis is similar to the presence of HA in the chemosensory organs of gastropods but is different than the sensory systems in vertebrates or arthropods. However, HA's widespread abundance throughout the rest of the CNS of Sepia is a similarity shared with gastropods, vertebrates, and arthropods. Its widespread use with differing functions across Animalia provokes questions regarding the evolutionary history and adaptability of HA as a transmitter.

[Plasmacytic skin infiltration in multiple myeloma]. / Myeloma multiplex kórlefolyása során megjelenô plazmasejtes bôrinfiltráció.

UNLABELLED: Authors present a case of a therapy-resistant multiple myeloma who developed plasmacytic skin infiltration in the course of the disease. AIM: To define characteristics of skin infiltrating plasma cells, which differentiate them from those cells residing in the bone marrow in order to contribute to a better understanding of the epidermoinvasion process. METHODS: Histidine decarboxylase is the only enzyme capable for histamine synthesis having significance in cell proliferation. Histidine decarboxylase was determined in skin samples and bone marrow slides by immunohistochemical procedures and in bone marrow cells using flow cytometry analysis. RESULTS: The histidine decarboxylase expression of plasma cells participating in skin invasion disappeared, while that of bone marrow plasma cells remained. CONCLUSIONS: Authors conclude that the histidine decarboxylase loss would serve as an evidence for the dedifferentiation of epidermoinvasive cells as being the result of fundamental changes in histamine metabolism. As extramedullary myeloma cells differ from those residing in the bone marrow, their therapeutical response might also be different.

High gastrin cell activity and low ghrelin cell activity in high-anxiety Wistar Kyoto rats.

Ghrelin is produced by gastric A-like cells and released in response to food deprivation. Interestingly, psychological stress also raises circulating ghrelin levels. This study compared plasma ghrelin levels in Sprague-Dawley (SPD) rats and high-anxiety Wistar Kyoto (WKY) rats. The two strains were also compared with respect to plasma gastrin, a gastric hormone with a pre- and postprandial release pattern opposite to that of ghrelin, and to the activity of the gastrin-dependent, histamine-forming ECL cells in the gastric mucosa. The rats were killed after being freely fed or after an over-night fast. The stomachs were weighed and tissue samples were collected for histological and biochemical analysis. Plasma ghrelin and gastrin levels were determined by RIA. While fasted SPD rats had higher plasma ghrelin levels than fasted WKY rats (P < 0.001), plasma ghrelin did not differ between freely fed rats of the two strains. Gastrin levels were higher in fed WKY rats than in fed SPD rats (P < 0.001). Despite the higher plasma gastrin level, the oxyntic mucosal histidine decarboxylase (HDC) activity (a marker of ECL-cell activity) in fed rats and the mucosal thickness did not differ between the two strains. In a subsequent study, rats were subjected to water-avoidance stress for 60 min, causing plasma gastrin to increase in WKY rats (P < 0.001) but not in SPD rats. In conclusion, high-anxiety WKY rats had lower circulating ghrelin and higher gastrin than SPD rats in both the fasted and fed state, while the ECL-cell activity (HDC activity) was only moderately affected.

Heat Resistance of Histidine Decarboxylase from Gram-Negative Histamine-Producing Bacteria in Seafood.

Precooking of tuna is a potential critical control point (CCP) in the commercial manufacturing of canned tuna. To assess the efficacy of precooking as a CCP, an understanding of the thermal properties of histamine-producing bacteria (HPB) and their histidine decarboxylase (HDC) enzymes is required. The thermal properties of many HPB have been determined, but the thermal resistances of the HDC enzymes are unknown. The purpose of this study was to determine the D- and z-values of selected HDC enzymes to evaluate the CCP of precooking during the canning process and provide scientific data to support U.S. Food and Drug Administration guidelines. HDC (hdc) genes from three strains each of Morganella morganii, Enterobacter aerogenes, Raoultella planticola, and Photobacterium damselae were cloned, expressed, and purified using the Champion pET Directional TOPO Expression System, pET100 cloning vector, and HisPur Cobalt resin. The heat resistances of all enzymes were compared at 50°C, and the D- and z-values from one strain of each HPB were determined at 50 to 60°C. To evaluate the heat inactivation of HDC enzymes during canned tuna processing, tuna tissue was inoculated with HDCs and heated to 60°C in a water bath set at 65 and 100°C. The D-values for the HDC enzymes from M. morganii, E. aerogenes, R. planticola, and P. damselae ranged from 1.6 to 4.1, 1.6 to 6.3, 1.9 to 4.3, and 1.6 to 2.9 min, respectively, at 50 to 60°C. The z-values for M. morganii, E. aerogenes, R. planticola, and P. damselae were 19.2, 18.0, 22.0, and 13.3°C, respectively. The HDCs from all HPB except E. aerogenes showed no significant activity after being heated to 60°C. The data generated in this study will help refine current guidelines for the thermal destruction of the HDC enzymes.

Development of a high-throughput assay to measure histidine decarboxylase activity.

Histamine is a well-known mediator of allergic, inflammatory, and neurological responses. More recent studies suggest a role for histamine and its receptors in a wide range of biological processes, including T-cell maturation and bone remodeling. Histamine serum levels are regulated mainly by the activity of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Despite the importance of this enzyme in many physiological processes, very few potent HDC inhibitors have been identified. HDC assays suitable for high-throughput screening have not been reported. The authors describe the development of a fluorescence polarization assay to measure HDC enzymatic activity. They used a fluorescein-histamine probe that binds with high affinity to an antihistamine antibody for detection. Importantly, they show that probe binding is fully competed by histamine, but no competition by the HDC substrate histidine was observed. The automated assay was performed in a total volume of 60 muL, had an assay window of 80 to 100 mP, and had a Z' factor of 0.6 to 0.7. This assay provides new tools to study HDC activity and pharmacological modulation of histamine levels.

Immunohistochemical detection of histidine decarboxylase in neoplastic mast cells in patients with systemic mastocytosis.

Synthesis of histamine in hematopoietic progenitor cells may be one of the earliest events in mastopoiesis. We therefore asked whether the key enzyme involved in histamine production, histidine decarboxylase (HDC), can be used as an immunohistochemical marker for the detection of immature neoplastic mast cells (MC) in patients with MC-proliferative disorders. To address this question, we examined bone marrow biopsy specimens in a cohort of 102 patients with mastocytosis using an antibody against HDC. Independent of the maturation stage of MC, the anti-HDC antibody produced clear diagnostic staining results in all patients with systemic MC disease examined including those with MC leukemia and MC sarcoma, in which MCs are particularly immature. In these patients, expression of HDC was reconfirmed at the messenger RNA level by reverse transcriptase polymerase chain reaction analyses performed with RNA of highly enriched CD117(+) MC. In summary, HDC is expressed in neoplastic MC in patients with systemic mastocytosis independent of the maturation stage of cells or the variant of disease. Histidine decarboxylase should therefore be considered as a new MC marker in the screen panel of antigens used to diagnose high-grade MC malignancies.

Involvement of MAP kinases in lipopolysaccharide-induced histamine production in RAW 264 cells.

Roles of mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-induced production of histamine in the mouse macrophage-like cell line RAW 264 were analyzed. Incubation of RAW 264 cells in the presence of LPS increased histamine levels in the conditioned medium in a concentration- and time-dependent manner. The levels of histidine decarboxylase (HDC) mRNA and the 74-kDa HDC protein were also increased at 4 to 8 h and 8 to 12 h, respectively. LPS elicited the phosphorylation of p44/42 MAP kinase, p38 MAP kinase, and c-Jun N-terminal kinase (JNK). The MAP kinase-Erk kinase 1 inhibitor U0126 (0.1-10 microM) suppressed the LPS-induced phosphorylation of p44/42 MAP kinase, and inhibited the LPS-induced production of histamine and expression of the HDC mRNA and 74-kDa HDC protein in a concentration-dependent manner. The JNK inhibitor SP600125 (3-30 microM) suppressed the LPS-induced phosphorylation of c-Jun, and inhibited the LPS-induced production of histamine and expression of the HDC mRNA and 74-kDa protein in a concentration-dependent manner. Combined treatment with U0126 (0.3 microM) and SP600125 (10 microM) inhibited the LPS-induced production of histamine additively. The p38 MAP kinase inhibitor SB203580 (0.1-10 microM) partially inhibited the LPS-induced production of histamine. These findings suggest that LPS increases histamine production in RAW 264 cells by inducing the expression of the 74-kDa HDC protein, and that the LPS-induced expression of HDC is up-regulated at the transcriptional level by MAP kinases, especially p44 MAP kinase and JNK.

ECL-cell carcinoids and carcinoma in patients homozygous for an inactivating mutation in the gastric H(+) K(+) ATPase alpha subunit.

A family with a missense variant of the ATP4A gene encoding the alpha subunit of the gastric proton pump (H(+) K(+) ATPase) has recently been described. Homozygous siblings were hypergastrinemic (median gastrin 486 pM) and had gastric tumours diagnosed at a median age of 33 years. In the current histopathological study, we further characterized the tumours found in the gastric corpus. The tumours had the histological appearance of carcinoids (NET G1 or G2) and were immunoreactive for the general neuroendocrine markers chromogranin A (CgA) and synaptophysin as well as the ECL-cell markers vesicular monoamine transporter 2 (VMAT2) and histidine decarbozylase (HDC). One of the tumours consisted of a NET G2 component, but also had a component with glandular growth, which morphologically was classified as an intestinal type adenocarcinoma. Many glands of the adenocarcinoma contained a large proportion of cells positive for neuroendocrine markers, especially the small vesicle marker synaptophysin and the cytoplasmic enzyme HDC. In conclusion, patients homozygous for an inactivating ATP4A mutation develop gastric ECL-cell carcinoids in their 3rd or 4th decade. The adenocarcinoma may be classified as neuroendocrine with ECL-cell differentiation.

Dedifferentiation of enterochromaffin-like cells in gastric cancer of hypergastrinemic cotton rats.

The role of enterochromaffin-like (ECL) cells in gastric carcinogenesis is not fully understood. Spontaneous tumours developing in hypergastrinemic female cotton rats have an adenocarcinoma phenotype, but numerous cells in the dysplastic mucosa as well as in the carcinomas are positive for neuroendocrine markers. In the present study of female cotton rats with 2 and 8 months' hypergastrinemia, the oxyntic mucosa of the stomach was examined histologically and immunolabelled for histidine decarboxylase (HDC) and pancreastatin, and hyperplastic and neoplastic ECL cells were evaluated by electron microscopy. These animals developed hyperplasia of the oxyntic mucosa in general and of the ECL cells in particular after 2 months and dysplasia and carcinomas after 8 months. The immunoreactivity of the ECL cells in the oxyntic mucosa was increased at 2 months and declined at 8 months. These histological changes were associated with progressive loss of secretory vesicles and granules in ECL cells. We suggest that ECL cells in hypergastrinemic cotton rats dedifferentiate with time and that the gastric carcinomas may develop from ECL cells.

Concentration of histamine in serum and tissues of the primary ductal breast cancers in women.

The aim of this study was to evaluate the concentration of histamine (HA) and the activities of their enzymes, namely histidine decarboxylase (HDC) and diaminooxydase (DAO) in 95 women with ductal breast cancer and in healthy women. The control group comprised 60 women without any pathological changes in their breasts, in whom mammoplasties were performed. In women with breast cancer the concentration of HA in serum was significantly higher than in healthy controls (9.1+/-3.2 vs. 5.9+/-3.1 nmol/l; P<0.001). The concentration of HA was significantly higher in neoplasmatic tissues of women with breast cancers than in unchanged tissues of healthy subjects in the control group (14.2+/-5.1 vs. 6.3+/-9.1 nmol/g; P<0.001). HDC activity was significantly elevated in cancerous tissues of women with breast cancer relative to unchanged tissues of healthy subjects (54.7+/-17.1 vs. 39.3+/-26.9 pmol/min per mg; P<0.01). However, the activity of DAO was significantly lower (14.0+/-0.4 vs. 36.1+/-9.7 pmol/min per mg; P<0.001) in neoplasmatic tissues than in normal tissues of healthy women. The adjacent healthy tissue of cancer revealed higher concentrations of HA than were found in unchanged tissues of healthy subjects (6.3+/-9.1 vs. 7.5+/-5.4 pmol/min per mg), but this difference did not reach statistical significance. The activity of HDC did not show any significant difference between the healthy tissues adjacent to cancer foci of women with breast cancer and normal tissues obtained from healthy subjects (39.3+/-26.9 vs. 34.5+/-24.3 pmol/min per mg). However, the activity of DAO was markedly lower than in unchanged tissues of healthy women in the control group (36.1+/-9.7 vs. 14.4+/-10.9 pmol/min per mg; P<0.001). The concentration of HA in cancerous tissues was significantly higher than in adjacent healthy tissues (14.2+/-5.1 vs. 7.5+/-5.4 nmol/g; P<0.001). The activity of HDC was significantly higher in cancerous tissues than in adjacent healthy tissues (54.7+/-17.1 vs. 34.5+/-24.3 pmol/min per mg; P<0.001), but there was no difference in the activity of DAO (14.0+/-6.4 vs. 14.4+/-10.9 pmol/min per mg). The significant elevation of HA concentration in cancerous tissues of women with the ductal breast cancers is caused by the increased synthesis and decreased inactivation of HA.

Immunoreactivity of gastric ECL and A-like cells in fasted and fed rats and mice.

The oxyntic mucosa of rat and mouse stomach harbors histamine-producing ECL cells and ghrelin-producing A-like cells. The ECL cells are known to be active when the circulating gastrin levels are elevated in response to food intake. The A-like cells are the main source of circulating ghrelin. In response to starvation, the circulating ghrelin is elevated as a hunger signal. The aim of the present work was to study the correlation between the immunoreactivities and cellular activities of the ECL cells and A-like cells. Rats were either fed or fasted for 48 h and mice for 24 h. Immunohistochemical examination with antiserum against chromogranin A-derived fragment pancreastatin revealed both the ECL cells and the A-like cells without a difference between fasted and fed animals. Histamine was limited to the ECL cells with no significant difference between fasted and fed animals. Histidine decarboxylase (HDC) immunoreactivity occurred predominately in the ECL cells of the fed, but not fasted, animals in which the HDC enzymatic activity in the oxyntic mucosa was higher than in fasted animals. Ghrelin immunoreactivity was increased in terms of intensity, but not cell density in fasted animals. Thus, the immunoreactivities of ECL cells and A-like cells might be affected by starvation.

Histidine decarboxylase and urinary methylimidazoleacetic acid in gastric neuroendocrine cells and tumours.

AIM: To study histidine decarboxylase (HDC) expression in normal and neoplastic gastric neuroendocrine cells in relationship to the main histamine metabolite. METHODS: Control tissues from fundus (n = 3) and corpus (n = 3) mucosa of six patients undergoing operations for gastric adenocarcinoma, biopsy and/or gastric surgical specimens from 64 patients with primary gastric neuroendocrine tumours (GNETs), as well as metastases from 22 of these patients, were investigated using conventional immunohistochemistry and double immunofluorescence with commercial antibodies vs vesicular monoamine transporter 2 (VMAT-2), HDC and ghrelin. The urinary excretion of the main histamine metabolite methylimidazoleacetic acid (U-MeImAA) was determined using high-performance liquid chromatography in 27 of the 64 patients. RESULTS: In the gastric mucosa of the control tissues, co-localization studies identified neuroendocrine cells that showed immunoreactivity only to VMAT-2 and others with reactivity only to HDC. A third cell population co-expressed both antigens. There was no co-expression of HDC and ghrelin. Similar results were obtained in the foci of neuroendocrine cell hyperplasia associated with chronic atrophic gastritis type A and also in the tumours. The relative incidence of the three aforementioned markers varied in the tumours that were examined using conventional immunohistochemistry. All of these GNETs revealed both VMAT-2 and HDC immunoreactivity, and their metastases showed an immunohistochemical pattern and frequency similar to that of their primary tumours. In four patients, increased U-MeImAA excretion was detected, but only two of the patients exhibited related endocrine symptoms. CONCLUSION: Human enterochromaffin-like cells appear to partially co-express VMAT-2 and HDC. Co-expression of VMAT-2 and HDC might be required for increased histamine production in patients with GNETs.

Recombinant expression of rat histidine decarboxylase: generation of antibodies useful for western blot analysis.

Histidine decarboxylase catalyses the formation of histamine, an important biological messenger. In spite of the essential biological functions exerted by histamine the knowledge about the mechanisms involved in the regulation of histidine decarboxylase is rather limited. This is most likely due to the limited supply of suitable tools, including highly specific antibodies. In the present study we describe the production and characterisation of specific antisera against rat histidine decarboxylase using recombinant protein synthesised in a bacterial expression system. The antisera were shown to effectively immunoprecipitate histidine decarboxylase activity in extracts of fetal rat liver as well as to detect the histidine decarboxylase protein by Western blot analysis of COS-7 cells expressing recombinant rat histidine decarboxylase. The results demonstrate the successful production of highly specific antisera to histidine decarboxylase which may become valuable tools in future studies of the structure and function of this enzyme.

Insulin receptor-related receptor messenger ribonucleic acid in the stomach is focally expressed in the enterochromaffin-like cells.

The insulin receptor-related receptor (IRR) is a member of the insulin receptor family. In contrast to the widespread expression of insulin receptor and insulin-like growth factor-I receptor messenger RNA (mRNA), the expression of IRR mRNA is highly restricted to the kidney and stomach. IRR mRNA in the kidney is focally expressed in the renal distal tubule cells. However, the cellular localization of IRR mRNA in the stomach remains to be elucidated. Here, we examined the cellular localization of IRR mRNA in the rat stomach by in situ hybridization. IRR mRNA in the stomach was abundantly localized in the basal third of the oxyntic glands of the fundic stomach. IRR mRNA in the stomach was colocalized with mRNA for histidine decarboxylase, a marker for the enterochromaffin-like (ECL) cells, indicating that the expression was restricted to ECL cells. ECL cells actively produce and store histamine, which is an important physiological stimulant of acid secretion from the parietal cells. The preferential localization of IRR mRNA in ECL cells suggests that the IRR plays an important role in the function of these cells.

The connections between the septum-diagonal band complex and histaminergic neurons in the posterior hypothalamus of the rat. Anterograde tracing with Phaseolus vulgaris-leucoagglutinin combined with immunocytochemistry of histidine decarboxylase.

The connections between nuclei of the septum-diagonal band complex and the clusters of histaminergic neurons in the posterior hypothalamic region were studied with a dual-labeling procedure in which anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin was combined with immunohistochemistry of histidine decarboxylase. Phaseolus vulgaris-leucoagglutinin was injected in the medial and lateral septal nuclei, and in various parts of the nuclei of the diagonal band of Broca. The fibers arising from the medial and lateral septal nuclei traverse the vertical limb of the diagonal band and, in part, join the medial forebrain bundle in the preoptic area. Other fibers descend diffusely through the lateral hypothalamus to the posterior hypothalamus, or course in a bundle of fibers ensheathing the fornix. The nuclei of the diagonal band project via the medial forebrain bundle and the diffuse pathway to the posterior hypothalamic region. All the nuclei of the septum-diagonal band complex, with the exception of the medial and lateral parts of the nucleus of the horizontal limb of the diagonal band, project to clusters of histaminergic neurons. These projections exhibit the following arrangement: along the axis lateral septal nucleus-medial septal nucleus-vertical limb of the diagonal band-medial part of the horizontal limb of the diagonal band, the septohypothalamic fibers decrease in density and distribute to fewer clusters of histaminergic neurons. Varicosities on the labeled fibers are formed in close proximity to the cell bodies and dendrites of the histaminergic neurons.

Blood flow, histamine content and histidine decarboxylase activity in rat skin grafts and their modification by cyclosporin-A.

1 An attempt has been made to monitor simultaneously the changes in blood flow, histamine content, histidine decarboxylase (HDC) activity and mast cell population in rat skin isografts and allografts. 2 The histamine content in rat skin allografts during rejection was decreased in contrast with our earlier observation in rabbits. 3 Although no definite correlation has been found between blood flow changes, histamine content or HDC activity, a reciprocal relationship appeared to exist between histamine content and HDC activity in the skin grafts, which might be a reflection of the immunosuppressive activity of this amine. 4 The immunosuppressive agent, cyclosporin-A (20 mg/kg daily) was able to prolong skin allograft survival and prevent the changes in histamine and HDC activity in allografts. 5 Possible implications of these findings are discussed in relation to current knowledge concerning the interactions between endothelial cells, lymphocytes and mast cells/basophils in graft rejection.