R.T. Cunningham

Influence of pathological progression on the balance between cellular and humoral immune responses in bovine tuberculosis

Studies of tuberculosis have suggested a shift in dominance from a T helper type 1 (Th1) towards a Th2 immune response that is associated with suppressed cell‐mediated immune (CMI) responses and increased humoral responses as the disease progresses. In this study a natural host disease model was used to investigate the balance of the evolving immune response towards Mycobacterium bovis infection in cattle with respect to pathogenesis. Cytokine analysis of CD4 T‐cell clones derived from M. bovis‐infected animals gave some indication that there was a possible relationship between enhanced pathogenesis and an increased ratio of Th0 [interleukin‐4‐positive/interferon‐γ‐positive (IL‐4+/IFN‐γ+)] clones to Th1 (IFN‐γ+) clones. All animals developed strong antimycobacterial CMI responses, but depressed cellular responses were evident as the disease progressed, with the IFN‐γ test failing to give consistently positive results in the latter stages. Furthermore, a stronger Th0 immune bias, depressed in vitro CMI responses, elevated levels of IL‐10 expression and enhanced humoral responses were also associated with increased pathology. In minimal disease, however, a strong Th1 immune bias was maintained and an anti‐M. bovis humoral response failed to develop. It was also seen that the level of the anti‐M. bovis immunoglobulin G1 (IgG1) isotype antibody responses correlated with the pathology scores, whereas CMI responses did not have as strong a relationship with the development of pathology. Therefore, the development and maintenance of a Th1 IFN‐γ response is associated with a greater control of M. bovis infection. Animals progressing from a Th1‐biased to a Th0‐biased immune response developed more extensive pathology and performed less well in CMI‐based diagnostic tests but developed strong IgG1 humoral responses.

Cell-specific Processing of Chromogranin A in Endocrine Cells of the Rat Stomach

The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine (b) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylase (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA291–319 and CGA316–321 immunostained D cells exclusively, whereas antisera raised against bCgA82–91 and CgA121–128 immunostained A-like cells and D cells. Antisera raised against CgA318–349 and CgA437–448 immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA291–319 immunostained D cells, and antisera against CgA351–356 immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations.

Serum neurone-specific enolase concentrations in patients with neurological disorders

A radioimmunoassay (RIA) has been developed for neurone-specific enolase (NSE) and used to measure serum levels in patients with a range of neurological disorders. Serum NSE levels were within the normal range in 21 patients with multiple sclerosis and 4 patients with Guillain-Barre syndrome. Normal serum NSE levels were also recorded in patients with motor neurone disease, anterior spinal thrombosis, multi-infarct disease, benign intracranial hypertension and peripheral neuropathy. However, two patients in coma, one as a result of encephalitis, the other due to subarachnoid haemorrhage (SAH) had elevated serum NSE. In the former, serum NSE levels appeared to predict a deterioration in clinical state, levels later returning to normal before an improvement in clinical condition. In the patient with SAH, levels were elevated on admission and remained elevated until death. Serum NSE levels may be of use in predicting outcome in patients with acute neurological disease.

Immunostaining for vasostatin I distinguishes between ileal and lung carcinoids.

Although chromogranin A (CgA) is a recognized marker of neuroendocrine tumours, little is known about the distribution of the CgA‐derived peptides, vasostatin (VST) I or II, in these tumours. Rabbit polyclonal antiserum was raised to a fragment of VST I and used to immunostain sections (5μm) of wax‐embedded tumour tissue. Immunoreactivity (IR) was detected using swine anti‐rabbit fluorescein secondary antibody and sections were viewed by fluorescence microscopy. Of 24 tumours from patients with lung carcinoids, one was weakly positive, while 23 of 26 ileal carcinoid tumours were immunoreactive. Metastatic deposits from patients with ileal carcinoids also tended to be immunoreactive (9/10). The difference in IR between lung and ileal carcinoid primary tumours did not appear to be related to the metastatic potential, since appendiceal tumours, which seldom metastasize, also tended to be immunoreactive (4/6) for VST I. The strongest IR was recorded in two patients with flushing as a result of ileal carcinoids; five other ‘flushers’ with ileal carcinoids were also immunopositive for VST I‐like IR. By contrast, patients with flushing as a result of lung carcinoids were immunonegative for VST. In conclusion, VST I‐like IR may assist in the identification of a secondary deposit from an unknown primary site. Copyright

Development of a radioimmunoassay for neurone specific enolase (NSE) and its application in the study of patients receiving intra hepatic arterial streptozotocin and floxuridine

A radioimmunoassay has been developed for neurone specific enolase (NSE) and used to measure serum NSE levels in patients with neuroendocrine and non-neuroendocrine tumours following intra hepatic arterial chemotherapy. Ten patients were studied, 7 receiving streptozotocin and floxuridine for neuroendocrine tumours and three receiving cisplatinum for non-neuroendocrine neoplasms. All ten patients had liver metastases. In patients with tumours of neuroendocrine origin, a significant increase in serum NSE was recorded within 24 h of therapy. Slight increases in serum NSE levels were also recorded in three patients with non neuroendocrine tumours. These increases may reflect lysis of neuroendocrine cells within the tumour. Raised levels in non-neuroendocrine tumour patients may reveal damage done to healthy neuronal and neuroendocrine cells during treatment. NSE may be a useful marker of the extent of cell death following chemotherapy.

Serum neurone-specific enolase levels in patients with neuroendocrine and carcinoid tumours.

We have examined concentrations of neurone-specific enolase (NSE) in sera from 18 patients with various neuroendocrine tumours, 26 patients with carcinoid tumours, 21 patients with non-neuroendocrine tumours and 37 control individuals. No statistically significant difference between the concentrations in patients with neuroendocrine tumours and patients with carcinoid tumours was found. However the NSE concentrations in patients with carcinoid and neuroendocrine tumours, when these two groups were combined, were significantly different from the patients with non-neuroendocrine tumours or the control individuals (P < or = 0.01). 38.5% of the patients with carcinoid tumours had raised NSE concentrations in serum; 55.5% of those with non carcinoid neuroendocrine tumours had raised concentrations. There appeared to be no correlation between the NSE concentrations and the extent of metastases.

TFF1 gene expression in human medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) is an uncommon tumour of calcitonin‐secreting C‐cells of the thyroid gland. This cancer represents an important potential model for the study of mechanisms of human epithelial cell transformation. Although recent studies have identified the gene involved in familial forms of MTC, little is known about the molecular pathogenesis of the sporadic variants of this tumour. The biological and prognostic significance of TFF1 expression, particularly in diverse human malignancies, suggests that the TFF1 protein could have a role in human neoplasia. Furthermore, in prostate cancer it has been demonstrated that TFF1 expression is closely associated with premalignant changes and neuroendocrine differentiation. In the present study, the expression of TFF1 was analysed in 18 human MTCs, comprising sporadic and familial tumours, C‐cell hyperplasia, and one case of lymph gland metastasis. TFF1 expression was also examined in the cultures of a human MTC‐derived tumour cell line (TT cell line). The results showed that ten sporadic tumours, three hereditary tumours (including C‐cell hyperplasia), and one lymph gland metastasis displayed TFF1 immunoreactivity. Indirect fluorescence immunocytochemistry and Western blotting revealed that the TFF1 protein was strongly expressed in the TT cells. Northern analysis revealed that tumours and TT cells expressed the TFF1 transcript. Although the function of TFF1 protein in the carcinogenesis of MTC remains to be elucidated, its expression in the majority of cases of both sporadic and hereditary tumours, metastatic tumours, and in C‐cell hyperplasia suggests that it may contribute to the pathogenesis of MTC.

Chromogranin A proteolysis to generate β-granin and WE-14 in the adenohypophysis during the rat oestrous cycle

Immunohistochemical analysis of the male and female rat adenohypophysis revealed that chromogranin A (CgA), beta-granin and WE-14 immunostaining was localised to follicle stimulating hormone (FSH) producing cells, while luteinizing hormone (LH) producing cells exhibited chromogranin A and beta-granin immunostaining. The intensity of chromogranin A, beta-granin and WE-14 immunostaining exhibited variation during the oestrous cycle; weak immunostaining was observed during proestrous and oestrous, corresponding with the lowest cellular concentration of luteinizing and follicle stimulating hormone. Chromogranin A and beta-granin immunostaining were similar in both the male and female (at dioestrous), however, a larger number of more intense WE-14 immunopositive cells were evident in the male adenohypophysis relative to the female at any stage of the cycle. The tissue and plasma concentrations of beta-granin and WE-14 immunoreactivity fluctuated throughout the oestrous cycle. Maximum and minimum beta-granin and WE-14 tissue concentration counterpoised the latent maximum and minimum plasma concentration. Chromatographic analysis of adenohypophysis extracts revealed the degree of chromogranin A proteolysis throughout the oestrous cycle; in contrast, plasma profiles consistently possessed a large chromogranin A-like immunoreactant. This data suggests that chromogranin A biosynthesis, proteolysis and the secretion of its derived peptides parallels that of the gonadotroph hormones throughout the oestrous cycle.

PC12 CELLS SHOW IMMUNOREACTIVITY TO A NUMBER OF PROTEINS AND PEPTIDES, INCLUDING VASOSTATIN

N-terminal chromogranin A (CGA) contains peptides with vasoinhibitory properties, called vasostatin I (VST) and II [CGA (1-76) and (1-113) in human and bovine; (1-128) in rat]. Three fragments of VST were synthesized and antisera raised: human CGA (68-76) (VST I) rat CGA (121-128) (VST II fragment 2), and bovine/human CGA (83-91) (VST II, fragment 3). Strong immunoreactivity was observed in PC12 cells with antisera to VST II, fragment 3, VST I, and neuron-specific enolase. Little or no immunoreactivity was observed using antisera to synaptophysin, whole molecule CGA, pancreastatin, protein gene product 9.5, somatostatin, pancreatic polypeptide, or with antibodies 875 and 876 to VST II, fragment 2. Most of the VST antisera cross-reacted, with a species of molecular weight, 61 kDa but one, 874, cross-reacted with two species of molecular weights, 7.2 and 12 kDa. Our results show the presence of N-terminally processed CGA in PC12 cells.