Comparison of immunomodulative effects of histamine-2 receptor antagonists in gastric cancer patients: Focus on the lymphoblastogenesis and cytotoxicity of peripheral blood mononuclear cells

A proposed mechanism of the immunomodulative effects of histamine-2 receptor antagonist (H2-RA) has been considered to be the inhibition of suppressor T-lymphocyte activity, an increase in interleukin-2 production of helper T-lymphocytes, and an enhancement of natural killer cell activity. Since there is a lack of comparative data about the immunomodulative effects of various H2-RAs, cimetidine, ranitidine and famotidine on peripheral blood mononuclear cells (PBMC), study of the comparison of the actions of H2-RA will be required. We compared the immunomodulative effect of each H2-RA on PBMC in patients with gastric cancer. DNA synthesis, cytotoxicity of PBMC against K562 cells and gastric cancer cell lines, and the levels of supernatant soluble interleukin-2 receptor (sIL-2R) were measured after the addition of each H2-RA, respectively. Increased suppressor cell activities were attenuated and restored to the levels of normal controls by the addition of cimetidine to H2-RA. Statistically significant lymphoblastogenesis and cytotoxicity against K562 cells were observed only in cimetidine-treated PBMC (P<0.05). Such effects were not observed in ranitidine- or famotidine-treated PBMC. Neither cimetidine- nor ranitidine-activated PBMC showed any significant cytotoxicity against gastric cancer cells. Significantly increased levels of sIL-2R were found in supernatants obtained from culture flasks treated with cimetidine or ranitidine and phytohemagglutinin (P<0.01). A significant correlation was found between the cytotoxicity of cimetidine- or ranitidine-treated PBMC and supernatant sIL-2R (P<0.05). In conclusion, the most strongly modulative substance among H2-RAs was cimetidine and the least modulative drug was famotidine. These results might be due to their structural differences. The immunological effects of H2-RA are unlikely to be mediated via specific interaction at the H2 receptor.     

Comparison of antiproliferative effects of 1-histamine-2 receptor antagonists,cimetidine, ranitidine,and famotidine,in gastric cancer cells

In the immune system, histamine is known to suppress cytotoxic T-lymphocytes and mitogen induced lymphocyte thymidine uptake, down-regulate some cytokines, and activate suppressor T-lymphocytes, and in the gastrointestinal system, histamine was reported to have trophic effects on gastrointestinal epithelial cells. Enhanced rates of cell proliferation by histamine are implicated in the pathogenesis of carcinogenesis. This study was designed since there is a lack of comparative data about the cell proliferations of histamine-2 receptor antagonist (H2-RA), cimetidine, ranitidine, and famotidine, in gastric cancer. KATO-III and AGS cell lines were used in this experiment. The concentrations of the histamine and cimetidine were 10⁻⁵, 10⁻⁸M, respectively and those of ranitidine and famotidine were 10⁻⁶-10⁻⁹M, respectively. Cell proliferation after drug treatment was evaluated by direct cell counting, [³H]thymidine incorporation, and MTT assay. Activities of ornithine decarboxylase (ODC), a rate limiting enzyme in polyamine synthesis, were measured after each drug treatment. Protein kinase A, a cAMP-dependent protein kinase system, was assayed using [α-³²p]ATP. Histamine showed statistically significant cell proliferating effects in a dose-dependent manner (P < 0.01), the maximal effect in 10⁻⁵M concentration. ODC activities were increased in accordance with the increment of cell numbers after histamine treatment. Cimetidine reversed the histamine-stimulated cell proliferation significantly, the maximal effect in 10⁻⁵M concentration (P < 0.01). Although ranitidine showed the tendency to attenuate the cell proliferation dose-dependently, but without statistical significance, famotidine did not show such an effect at all. cAMP-dependent protein kinase activities were significantly increased following 10⁻⁵M histamine treatment, also reversed significantly by cimetidine co-administration (P < 0.01). Beneficial clinical outcomes could be anticipated from cimetidine treatment in patients with gastric cancer by anti-proliferating effects against gastric cancer cells. These effects of H2-RA are likely to be mediated by specific interactions at the H2-receptor.     

Controlled clinical trial of treatment with cimetidine for non-ulcer dyspepsia

The effect of cimetidine (1 g daily) and placebo was studied in a controlled clinical trial comprising 50 patients with non-ulcer dyspepsia in whom an organic abnormality responsible for the dyspeptic symptoms was not disclosed by a standardized and extensive examination programme. Reduction of symptoms occurred in 13 (54%) out of 24 patients treated with cimetidine and in 16 (62%) out of 26 treated with placebo. The difference was insignificant, as were the alterations in the individual dyspeptic symptoms between the groups. Only 6 patients (25%) on cimetidine and 8 (31%) on placebo treatment had a total relief of symptoms. Of these, all cimetidine-treated patients remained free from symptoms during the successive 3-month observation period, while the dyspeptic symptoms relapsed in 3 (38%) placebo-treated patients. Subsequent resumption of placebo treatment reduced the symptoms in all 3 patients, but only one became free from symptoms. Cimetidine does not seem to be superior to placebo in the treatment of non-ulcer dyspepsia in patients without any previous history of ulcer or without any sign on endoscopy of an active or previous ulcer disease.     

CD83+CCR7− Dendritic Cells Accumulate in the Subepithelial Dome and Internalize Translocated Escherichia coli HB101 in the Peyer’s Patches of Ileal Crohn’s Disease

Recurrent Crohn’s disease originates with small erosions in the follicle-associated epithelium overlying the Peyer’s patches. Animal studies have illustrated mucosal immune regulation by dendritic cells located in the subepithelial dome. The aim of this study was to characterize the dendritic cells at this specific site in patients with Crohn’s disease. Ileal tissues were obtained after surgery performed on Crohn’s patients; ileal samples from noninflammatory bowel disease and ulcerative colitis served as standard and inflammatory controls, respectively. Flow cytometry of isolated intestinal mononuclear cells showed a larger subset of dendritic cells in Crohn’s samples compared with controls. This finding was corroborated by confocal microscopy, showing enhanced infiltrates of cells positive for the dendritic cell markers, DC-SIGN⁺ and CD83⁺, in the subepithelial dome. Moreover, the CD83⁺ cells in Crohn’s tissues showed reduced expression of the lymph node migratory receptor, CCR7, possibly contributing to the high numbers of dendritic cells. After exposure to nonpathogenic Escherichia coli in Ussing chambers, dendritic cells in the subepithelial dome of Crohn’s disease demonstrated increased co-localization with translocated bacteria. Immunohistochemical results revealed that DC-SIGN⁺ cells in Crohn’s tissues were found to express toll-like receptor 4 and produce tumor necrosis factor-α. In conclusion, nonmigrating dendritic cells that accumulate in the subepithelial dome and internalize nonpathogenic bacteria may be important for the onset and perpetuation of mucosal inflammation in Crohn’s disease.     

Cimetidine for anastomotic ulcers after partial gastrectomy. A randomized controlled trial.

In a randomized double-blind multicenter trial, 15 outpatients with endoscopically proved anastomotic ulceration after Billroth I or Billroth II partial gastrectomy received cimetidine, 1 g daily over eight weeks, or a placebo. All patients also received antiacid. The ulcer healed completely in all seven cimetidine-treated patients and in one of the eight placebo-treated patients (P less than 0.01). Ulcers not healed during the double-blind phase of the trial were all subsequently healed on open cimetidine treatment. There was a trend toward improvement of daytime symptoms in favor of cimetidine (P = 0.06), and nighttime symptoms were significantly relieved during the initial four weeks of cimetidine treatment P = 0.02). We conclude that cimetidine, 1 g daily, promotes healing of anastomotic ulcers after partial gastrectomy.     

Oral administration of non-absorbable delayed release 6-mercaptopurine is locally active in the gut,exerts a systemic immune effect and alleviates Crohn’s disease with low rate of side effects: results of double blind Phase II clinical trial

Therapy for Crohn’s disease (CD) with thiopurines is limited by systemic side effects. A novel formulation of fixed-dose, delayed-release 6-mercaptopurine (DR-6MP) was developed, with local effect on the gut immune system and minimal absorption. The aim of this study was to evaluate the safety and efficacy of DR-6MP in patients with moderately severe CD compared to systemically delivered 6-mercaptopurine (Purinethol). Seventy CD patients were enrolled into a 12-week, double-blind controlled trial. The primary end-point was the percentage of subjects with clinical remission [Crohn’s Disease Activity Index (CDAI) < 150] or clinical response (100-point CDAI reduction). Twenty-six (56·5%) and 13 (54·2%) subjects from the DR-6MP and Purinethol cohorts, respectively, completed the study. DR-6MP had similar efficacy to Purinethol following 12 weeks of treatment. However, the time to maximal clinical response was 8 weeks for DR-6MP versus 12 weeks for Purinethol. A higher proportion of patients on DR-6MP showed clinical remission at week 8. A greater improvement in Inflammatory Bowel Disease Questionnaire (IBDQ) score was noted in the DR-6MP group. DR-6MP led to a decrease of CD62⁺ expression on T cells, implying a reduction of lymphocyte adhesion to site of inflammation. DR-6MP was safer than Purinethol, with significantly fewer adverse events (AEs). There was no evidence of drug-induced leucopenia in the DR-6MP group; the proportion of subjects who developed hepatotoxicity was lower for the DR-6MP. Non-absorbable DR-6MP is safe and biologically active in the gut. It is clinically effective, exerting a systemic immune response with low systemic bioavailability and a low incidence of side effects.     

Infiltration of CD19+ plasma cells with frequent labeling of Ki-67 in corticosteroid-resistant active ulcerative colitis

Abnormalities in humoral immunity are implicated in the pathogenesis of ulcerative colitis. However, the detailed mechanisms of B-cell activation in the locale remain unaccounted for. We analyzed ulcerative colitis from the standpoint of lymphocytic expansion in the loco. Intestinal specimens obtained at surgery from 30 patients with ulcerative colitis treated with corticosteroids and 15 with Crohn’s disease were analyzed by immunohistochemistry and flow cytometry. Ulcerative colitis was characterized by a diffuse distribution of Ki-67⁺ small round cells particularly in the ulcer base (that were CD19⁺ and CD20^–), with a significant number of them also CD138⁺. Immunoelectron microscopy for CD19 revealed an abundance of rough endoplasmic reticulum in the cytoplasm. These indicated that they are of immature plasma lineage cells. By contrast, plasma cells in Crohn’s disease were negative for CD19, and the labeling for Ki-67 was infrequent, showing mature phenotype. Flow cytometry revealed an occurrence of CD19⁺ and CD20^– cells in ulcerative colitis but not in Crohn’s disease. The labeling index of Ki-67 among CD19⁺ plasma cells was positively correlated with the clinical activity of ulcerative colitis. High labeling of Ki-67 in CD19⁺ plasma cells is specific for active ulcerative colitis that was resistant to medical treatment by corticosteroids.     

Effect of the Immune Modulators BM 12.531 (Azimexone) and BM 41.332 on the Subsets of T-Lymphocytes in Mice

Abstract

The immunomodulating 2-cyanaziridine derivatives BM 12.531 (azimexone) and BM 41.332 have no effects on the total amount of T-lymphocytes in the spleen of mice but ingrease dose-dependently the percentage of Ly1^?-, 2⁺, 3⁺ T-lymphocytes (killer/suppressor) and decrease the percentage of Ly1⁺, 2^?-, (helper) cells. These investigations were carried out by means of specific monoclonal antibodies and fluoreqcence-activated cell sorting. The increase in Ly2⁺-cells is mainly due to increased suppressor activity.     

The effect of cimetidine on antibody synthesis in vitro and in vivo

The effects of the H₂-receptor antagonist cimetidine on the antibody response of murine spleen cells to sheep red blood cells (SRBC) has been examined in vitro and in vivo. Studies failed to demonstrate any detectable effect of the drug in vitro over a wide range of concentrations (10^-12 to 10^-4M). In vivo, cimetidine stimulated IgM but not IgG responses. This effect was more marked on the secondary rather than the primary response. Low doses of cimetidine (0.5–10 mg/kg) enhanced the secondary IgM response better than higher concentrations (50–100 mg/kg).     

Antimetastatic effect of cimetidine on mice bearing a C3H mouse mammary adenocarcinoma: survival and lymphocyte function studies

It has been reported that treatment with cimetidine, a histamine H₂-receptor antagonist, increased survival and decreased the number of lung metastases in mice bearing the Lewis Lung carcinoma [29]. It was suggested that this effect was due to the ability of cimetidine to block histamine activation of suppressor lymphocytes and hence allow host defence mechanisms to inhibit tumour growth. In the present studies, C3H/He mice were implanted with a C3H mouse mammary adenocarcinoma on Day 0. This tumour metastasizes to the lungs in 30–50 days. Primary tumours were ablated with X-rays when they had grown to about 0·2g and animals were given drinking water with or without cimetidine (10 mg ml^–1) until the end of the experiment. Cimetidine reduced the number of mice dying from metastatic disease from 7/15 (controls) to 3/13. Cimetidine treatment also prolonged survival of mice that did succumb to metastatic disease by about 12 days. The response of spleen lymphocytes to the mitogens phytohaemagglutinin and lipopolysaccharide was assessed in vitro by uptake of ³H-thymidine 0, 16, 45 and 58 days after tumour implantation. Lymphocyte responsiveness was depressed by tumour burden. The influence of cimetidine treatment was equivocal being dependent upon time after tumour implantation and dose of mitogen. In this mouse-tumour system, the mechanism of the antimetastic effect of cimetidine is different from that previously suggested [29].     

A new T-lymphocyte cloning assay for detection of in vivo mutations in the human hypoxanthine-guanine phosphoribosyltransferase gene

The X-linked hypoxonthine-guanine phosphoribosyltransferase (hprt) gene is a target of analyses of in vivo mutation frequencies in circulating T-lymphocytes. We established a novel, accessory cell-free cloning method of T-lymphocytes with a hprt mutation by a combined use of recombinant interleukin-2, conditioned medium from activating T-lymphocytes and culture plates coated with anti-CD3 monoclonal antibody. Using the method, we examined mutation frequencies of the hprt gene in T-lymphocytes from six healthy individuals, nine patients with colon cancer including two patients from different families with hereditary nonpolyposis colon cancer and six cancer-free relatives of the patients. In six healthy individuals, the mean cloning efficiency and mutation frequency (MF) of the hprt gene in T-lymphocytes were 0.51 ± 0.28 and 9.4 ± 7.5 × 10⁻⁶, respectively. These data were similar to the reported values. The mean MFs in the nine colon cancer patients (10.6 ± 7.3 × 10⁻⁶) were not significantly different from those of the 12 cancer-free individuals (11.6 ± 9.4 × 10⁻⁶). The correlation between mutation frequencies and age of the individuals was significant regardless of the presence or absence of cancers. The single-strand conformation polymorphism analyses of nested RT-PCR products of hprt mRNA were done in 33 mutant clones from five members of a family of which MF values were high. All the analyzed mutant clones show a genetic aberration in the coding region of the hprt gene. At least 28 of 33 mutants were independent. Our method provides a new versatile tool for in vivo analysis for mutations of the hprt gene. Environ. Mol. Mutagen. 30:31–39, 1997 © 1997 Wiley-Liss, Inc.     

Vascular studies with histamine in vitro

In vitro studies to analyse the pharmacology of histamine-induced dilatation of resistance vessels in rat hindquarters have been made. Histamine caused dose-dependent dilatation of resistance vessels over the concentration range 10^–9 to 10^–6 mol. Responses to histamine were antagonized by cimetidine but not by mepyramine. Dimaprit also caused vasodilatation. Responses to dimaprit were inhibited by cimetidine 10^–6 to 10^–5 M. A pA₂ of 6.43 (6.11–6.75, 95% confidence limits) was calculated for cimetidine in the resistance vessels of the hindquarters.     

The kinetics of cell surface receptor expression in children perinatally exposed to polychlorinated biphenyls

Exposure to polychlorinated biphenyls (PCBs) during pre-natal and early life can alter normal immune system development. Blood specimens from newborns, 6-, and 16-month-old infants were collected in the Michalovce and Svidnik/Stropkov districts, areas with, respectively, high and low environmental PCB contamination, and lymphocyte receptor expression was evaluated by multi-color flow cytometry. The results indicate that the percentage of lymphoid dendritic cells (DC) and naïve/resting T-lymphocytes were significantly increased at 6-months in Michalovce as compared to the same cell types in cord blood samples (p?<?0.001), whereas natural regulatory T-lymphocytes and suppressor inducer T-lymphocytes were reduced (p?<?0.001). Overall, a positive linear correlation of terminally differentiated effector memory (TEM) T-lymphocyte population with age, but a negative linear correlation for myeloid DC from birth to 6-months in both regions were found. Michalovce samples indicated significantly higher expression of memory T-lymphocytes (birth, 6^th, and 16^th month), TEM T-lymphocytes (birth and 6^th month), and lymphoid DC (6^th month) compared to the Svidnik/Stropkov regions. After adjustment for relevant covariates, such as maternal age, parity, season of birth, breastfeeding, birth weight, and gender, the myeloid DC, suppressor inducer T-lymphocytes, truly naïve helper/inducer T-lymphocytes, and TEM T-lymphocytes remained significantly different between districts in cord blood samples. The multivariate analysis models for 6- and 16-month samples showed district differences in all cellular determinants, except for lymphoid DC and macrophage-like cells. This study provides the first evidence that pre-natal and early post-natal exposure to PCBs affects the dynamics of cell surface receptor expression on lymphoid DC and DC-like cells, suggesting impaired immunologic development following pre-natal and early post-natal PCB exposure.     

The effect of histamine2 and muscarine receptor antagonists on plasma levels of parathyroid hormone and calcitonin

Summary Long-term administration of cimetidine, a histamine₂ receptor antagonist, has been reported to normalize elevated parathyroid hormone (PTH) concentrations in patients with secondary [1] and primary hyperparathyroidism [2] and even to improve the clinical symptoms. We have compared the effect of cimetidine and pirenzepine on PTH and calcitonin (CT) plasma levels in a short-term trial on patients with secondary hyperparathyroidism. After cimetidine a significant effect on PTH was seen within 30 min lasting 30 min and after pirenzepine, within 60 min and lasting 60 min. The effect on CT was only significant after cimetidine.     

Distinctive Pattern of Cytokine Production and Adhesion Molecule Expression in Peripheral Blood Memory CD4+ T Cells from Patients with Active Crohn’s Disease

An expansion of both circulating and intestinal lamina propria CD4⁺CD45RO⁺ T cells has been described in patients with Crohn’s disease. We studied both the cytokine profile and the expression of adhesion molecules on this T-cell subset. Peripheral blood CD4⁺CD45RO⁺ T cells from patients with Crohn’s disease (n=45) were assessed by flow cytometry and RT-PCR methods. The cytokine profile was also measured in intestinal lamina propria from seven patients. They were classified according to the CDAI and the results were compared with those of patients with ulcerative colitis (n=21) and noninflammatory intestinal conditions (n=15), and healthy controls (n=39). The mean percentage of circulating CD4⁺CD45RO⁺ T cells producing intracellular TNF was higher in active than in inactive Crohn’s disease patients (p < 0.001), active (p = 0.49) and inactive ulcerative colitis (p = 0.019), and healthy controls (p =0. 017). TNF expression correlated with CDAI (p < 0.001). An increased expression of intracellular IL-2, IL-6, and IL-10 in active Crohn’s disease patients was also found. CD62L was downregulated in active Crohn’s disease patients while no differences were observed in CD49d and CD11a expression. Lamina propria CD4⁺CD45RO⁺ T cells from active Crohn’s disease lesions showed an increased intracellular staining of TNF, IFN-γ, and IL-10. Both peripheral and intestinal mucosa CD4⁺CD45RO⁺ T cells from active Crohn’s disease patients show an increased production of TNF. In addition, the circulating CD4⁺CD45RO⁺ T-cell subset expresses a pattern of adhesion molecules that promotes homing to extranodal lymphoid tissues. This T-cell subset may play a relevant role in the immunopathogenesis of Crohn’s disease.Dr. García de Tena and Dr. Manzano are joint first authors     

The cellular composition of granulomas in mesenteric lymph nodes from patients with Crohn's disease

Summary The immunohistochemical findings in granulomatous lymphadenitis in patients with Crohn's disease are presented and compared with conventional light microscopic findings. The cellular composition of the granulomas in mesenteric lymph nodes was examined with a broad panel of monoclonal and polyclonal antibodies directed to B-cells, T-cells, monocytes/macrophages, dendritic reticulum cells, HLA-DR antigens and the transferrin receptor. The centre of the granulomas contains OKIa⁺, OKM1⁺, OKT9⁺, DRC^–, To5^– epithelioid cells and giant cells and OKT3⁺ lymphocytes. In general, the majority of the small lymphocytes within the granulomas expresses the OKT3⁺, OKT4⁺ Leu 3a⁺ phenotype which points toward T-helper cells. Fewer OKT3⁺ OKT8⁺ T suppressor/cytotoxic cells are observed. At the periphery of the granulomas a lymphocytic corona composed of BA1⁺, B1⁺ B lymphocytes may be present.From these findings it can be concluded that the granulomas in mesenteric lymph nodes from patients with Crohn's disease are composed of centrally located T-lymphocytes and of epithelioid cells which are of monocyte/macrophage origin and have the characteristics of antigen-presenting cells.     

Immune deficiency in HIV-1 infection: novel therapeutic approaches targeting innate and adaptive responses

HIV-1 is a lentivirus capable of infecting CD4⁺ T-lymphocytes and CD4-expressing innate immune cells. Infection with HIV-1 leads to rapid and early depletion of mucosal CD4⁺ T-cells, and to the establishment of viral reservoirs that are resistant to the most potent antiretroviral therapy currently available. Commonly observed virus-induced adaptive immune defects consist of weak-to-absent HIV-specific CD4 T-cell responses and of inappropriate or inefficient CD8⁺ cytotoxic T-lymphocyte activity. Moreover, the virus establishes early and long-lasting deficits in innate immunity characterized by reduced numbers or/and disrupted functions of antigen-presenting cells, natural killer cells and natural-kiiller T-lymphocytes. Therapeutic approaches have long been oriented toward restoration of adaptive immunity in HIV-1 patients. This is exemplified by the use, in antiretrovial treated subjects, of interleukin-2 to increase and expand CD4⁺ lymphocytes, and of structured treatment interruptions or therapeutic vaccination to restore HIV-specific responses. More recently, approaches aimed at correcting the deficits in innate immune responses have been explored. The most advanced of these strategies include synthetic immunomodulators targeting antigen presentation and human recombinant cytokines capable of regulating the functions of natural-killer cells. Today, in addition to the combined use of different classes of antiretrovirals, a highly active immune therapy, with components targeting both innate and adaptive responses, appears to be absolutely necessary to formulate immune control of the virus. Current and future clinical protocols will eventually define the timing, composition and formulations of combined HIV-specific and nonspecific immunotherapy that could be safely administered to HIV patients to restore immune homeostasis.     

Congenital cytomegalovirus: Immunological alterations

Immunological studies on 29 children with congenital cytomegalovirus (CMV) infection were performed. Two groups of patients were considered according to the presence or absence of clinical symptoms. Asymptomatic patients had increased numbers of natural killer (NK) cells (CD 16⁺), activated NK cells (CD16⁺/ HLA-DR), and activated suppressor/cytotoxic cells (CD8⁺/HLA-DR⁺/TAC). In addition, cells from this group of patients showed increased NK activity against fibroblasts infected with CMV and a significant proliferative response to CMV antigens. In contrast, cells from symptomatic patients had reduced NK activity against fibroblasts infected with CMV and a defective proliferative response to CMV antigens. In both groups, the number of total T, T-helper, and T-suppressor/cytotoxic cells was normal, as was the ratio of helper/suppressor. The reported differences in the immunological parameters between the asymptomatic and symptomatic patients suggest that cell-mediated immunity may play an important role in determining the outcome of the infection.     

Diagnosis of chronic myeloproliferative neoplasms with special emphasis on early stages

In the interdisciplinary approach to the diagnosis of chronic myeloproliferative neoplasms (MPN), bone marrow (BM) histopathology, clinical findings and molecular genetic abnormalities play a pivotal role. The recognition of the early stages of MPN requires long-term follow-up studies, including sequential BM examinations in combination with biological tests and the JAK2 V617F mutation status. Based on these rationales, the early stages of MPN, including polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), are described with corresponding haematological data. With reference to relevant complications (thromboembolic and haemorrhagic events and myelofibrotic transformation) and ensuing therapeutic options, the diagnostic value of a proper evaluation of the initial BM biopsies can not be overemphasized. Pre-polycythaemic stage PV, which often presents with a low but increasing haemoglobin level at onset, can be diagnosed from a positive JAK2 V617F mutation status, the characteristic BM features of panmyelosis (trilineage increase in erythro-, granulo- and megakary-opoiesis) and a serum erythropoietin level below the normal range. Early manifestations of ET can be recognized by lowering the platelet count (>600 x 10⁹/litre) to 450 × 10⁹/litre, BM morphology (predominance of large to giant megakaryocytes without abnormalities of maturation), as well as relevant complications (haemorrhage and vascular events). Histopathological characteristics and accompanying clinical features play a distinctive role in the establishment of pre-fibrotic early PMF. Due to their significant differences in progression to myelofibrosis and outcome, an important issue is the discrimination between pre-fibrotic PMF, which frequently presents with thrombocytosis, and ET. Up-to-date diagnostic criteria, originally established by the World Health Organization (WHO) and recently revised by a panel of experts, should be generally applied and validated in prospective clinical trials to achieve a consensus-based working diagnosis.     

A method for isolation and culture of lymphocytes from endoscopic biopsies

Human small and large intestinal lamina propria lymphocytes have been successfully prepared from endoscopic biopsies by a combined enzymatic and mechanical method which gives higher yields of viable mucosal lymphocytes than previously reported, despite the small size of the biopsy samples. Viability of the cells was demonstrated by dye exclusion and they could be satisfactorily maintained in short-term culture. Phytohaemagglutinin-P (PHA-P) transformation characteristics of intestinal lymphoid cells and those of peripheral blood were studied in 20 patients with Crohn's disease and 10 control subjects. Peripheral blood lymphocytes were separated according to this technique, no decrease in viability being observed when compared to a standard Ficoll-Hypaque gradient technique. Endoscopically abnormal (EA) and endoscopically normal (EN) Crohn's tissue showed significantly different responses to PHA-P (P<0.001), EA tissue lymphocytes giving lower blastogenic responses.