Skin biopsies for early diagnosis and prognosis of graft-versus-host disease in recipients of allogeneic stem cells from blood or bone marrow.

A total of 61 patients with haematological malignancies were randomised either to allogeneic transplantation with blood stem cells (BSC) or bone marrow (BM), of whom 37 patients gave their consent to participate in a skin biopsy trial. Skin biopsies were performed before and after transplantation. The main objective was to assess whether biopsies of normal and affected skin from patients allografted with BSC showed a different histopathological and immunohistochemical pattern as compared to biopsies taken from patients allografted with BM. In addition, we wished to clarify whether sequential skin biopsies could be of prognostic value with regard to graft-versus-host disease (GVHD). Biopsies from normal or affected skin in BSC allografted did not disclose a different pattern as compared to BM transplants. Biopsies taken before the outbreak of acute and chronic GVHD showed no substantial differences between the groups. Irrespective of the type of allograft, the immunohistochemical picture of affected skin consistent with acute GVHD was dominated by a significantly higher number of T-lymphocytes (CD8+). Biopsies from normal skin before the outbreak of GVHD had no predictive value with regard to the development of acute or chronic GVHD. Immunohistochemistry is of supplementary help in distinguishing changes caused by cytotoxic agents from those caused by acute GVHD.

Effect of tumour size on the in vivo growth inhibition of human colon carcinoma cells (HT-29) by colon mitosis inhibitor.

We have previously shown that the colon mitosis inhibitor (CMI) suppresses the growth of transplanted HT-29 human colon carcinoma cells by approximately 40%. However, this effect declined along the time-course, as the inoculums progressively grew larger. In the present work we designed a test to assess the effectiveness of CMI as a function of tumour size. After ranking the terminal tumours by ascending size in the control group and the CMI group the growth inhibition was calculated at each rank position. The observed negative correlation between control tumour size and CMI inhibition (r = -0.94, p < 0.001) clearly demonstrated decreased growth inhibition with increased tumour size. Consequently, a retrospective analysis of the smallest and slowest growing tumours showed a profound growth inhibition (72-81%, p < 0.008), whereas a similar analysis of the large and fast growing tumours revealed no significant CMI effect. The increased CMI effect among slow growing tumours was apparently not associated with increased susceptibility to CMI in a subset of slow growing cells because the slow growing subclone HT-29A4 did not show increased CMI effect. Furthermore, HT-29A4 displayed a similar tendency of decreased CMI effect with increased tumour size (r = -0.70, p < 0.001). Interestingly, the same tendency of increased growth inhibitory effect on smaller tumours was also seen with retinoic acid and difluoromethylornithine (r = -0.96, p < 0.001). The apparent enhanced responsiveness among small tumours underlines the importance of early chemoprevention and chemotherapy.

Uveal melanomas with optic nerve extension: report of two cases diagnosed by transvitreal biopsy, one of them with a multicentric tumour.

PURPOSE: To report two cases of invasive uveal melanomas, one of which showed multifocality. METHODS: Clinical examination, ultrasonography, colour Doppler analysis, cytological and histopathological evaluations. RESULTS: Transvitreal biopsy (case 1) or fine needle aspiration biopsy (FNAB) (case 2) revealed malignant melanomas in both patients. Light microscopy and immunohistochemical examinations substantiated the diagnosis of mixed cell type melanomas. In addition, one patient had a multifocal melanoma with papilloedema and colour Doppler findings suggestive of optic disc involvement. CONCLUSION: Transvitreal biopsy for histology or cytology is a reliable procedure to obtain an accurate diagnosis without delay of a lesion adjacent to the optic nerve head. In our two cases the biopsy findings led to enucleation.

Neuronal cell death in the visual cortex is a prominent feature of the X-linked recessive mitochondrial deafness-dystonia syndrome caused by mutations in the TIMM8a gene.

The Mohr-Tranebjaerg syndrome (MIM 304700) and the Jensen syndrome (MIM 311150) were previously reported as separate X-linked recessive deafness syndromes associated with progressive visual deterioration, dystonia, dementia, and psychiatric abnormalities. In the most extensively studied Norwegian family, the Mohr-Tranebjaerg syndrome was reported to be caused by a one-basepair deletion (151delT) in the deafness/dystonia peptide (DDP) gene at Xq22. This gene has been renamed TIMM8a. We identified a stop mutation (E24X) in the TIMM8a gene segregating with the disease in the original Danish family with the Jensen syndrome, which confirms that the two disorders are allelic conditions. We also report abnormal VEP examinations and neuropathological abnormalities in affected males from the two unrelated families with different mutations. The findings included neuronal cell loss in the optic nerve, retina, striate cortex, basal ganglia, and dorsal roots of the spinal cord. The demonstration of mitochondrial abnormalities in skeletal muscle biopsies in some patients is compatible with the suggestion from recent research that the TIMM8a protein is the human counterpart of an intermembrane mitochondrial transport protein, Tim8p, recently characterized in yeast. The clinical and neuropathological abnormalities associated with mutations in the TIMM8a gene support that this X-linked deafness-dystonia-optic neuropathy syndrome is an example of progressive neurodegeneration due to mutations in a nuclear gene necessary for some, yet unknown mitochondrial transport function. We recommend sequencing the TIMM8a gene, thorough ophthalmological examination, and measuring visual evoked potentials in clinically suspected male patients with either progressive hearing impairment, dystonia, or visual disability in order to establish an early diagnosis and provide appropriate genetic counselling.

Novel lymphocyte growth-inhibiting tripeptide: N-acetyl-glu-ser-GlyNH(2).

The limited and predetermined number of cells that constitutes an organ or specialized cell population is to all appearances regulated according to a negative feedback principle involving growth inhibitors with sufficient tissue specificity. To find growth-inhibiting factors in lymphoid cells, we followed established purification procedures and assays. We found a single-peak fraction in water extracts of dog spleen homogenates that inhibited proliferation of Molt (T cell) lymphoma cells at low concentrations in vitro, with no significant effect on a B cell lymphoma cell line (Ramos). C-terminal amino acid sequencing and MS analysis showed the factor to be a tripeptide: N-acetyl-Glu-Ser-GlyNH(2). Treatment with a synthetic tripeptide with the structure N-acetyl-Glu-Ser-GlyNH(2) decreased the number of cell doublings of Molt cells. The peptide also delayed cell flux at the G(2)-M transition of the cell cycle, while incorporation of tritiated thymidine was not altered at the examined time points in this cell line. However, DNA synthesis in PPD-stimulated normal human lymphocytes was significantly inhibited and with a bell-shaped dose-response curve.

An endogenous melanocyte-inhibiting tripeptide pyroGlu-Phe-GlyNH2 delays in vivo growth of monoclonal experimental melanoma.

The melanocyte-inhibiting tripeptide (MTP) pyroGlu-Phe-GlyNH2 is present in tissue cultures of non-transformed melanocytes and melanoma cells and influences melanocyte growth in vitro. The objective of the present study was to investigate a possible effect of MTP on the in vivo growth of B16A2, a monoclonal experimental melanoma. The B16A2 clone was established by the limited dilution technique. It has a reduced DNA content and displays slower growth both in vivo and in vitro compared to the parent cell line (B16). B16A2 cells were injected subcutaneously into hairless mice at four sites (300 000 cells in 0.25 ml buffer/site). MTP was given by i.p. injection 3 times a week at two concentrations (1 pmol and 1 nmol/animal). The control animals received the equal volume of solvent. The animals were sacrificed 1 and 2 weeks after tumour transplantation, and all tumours were weighed. One week after transplantation, the animals who received 1 pmol MTP had fewer tumours and a reduced tumour load. Two weeks after the transplantation, the differences between control and treated animals were no longer observed. The results indicate that MTP temporarily delays in vivo tumour growth.

Change in the extent of colonoscopic and histological involvement in ulcerative colitis over time.

OBJECTIVE: Colonoscopy has replaced barium enema as the method for determining the extent of disease in patients with ulcerative colitis (UC). Normally, the extent of disease is determined by direct visualization of the mucosa, but biopsies are also used with increasing frequency. Very little is known about the extent to which these two ways of assessing the extent of disease are correlated and whether the correlation differs over time. The aim of this study was to determine the changes in extent of disease assessed by direct visualization and by histological examination of the mucosa at the time of diagnosis and after 1 yr of follow-up in a cohort of incident cases of UC patients. METHODS: All new cases of UC in a defined population were identified during a 4-yr period (496 patients). Of these, 384 patients (78%) were available for follow-up and were subjected to a second colonoscopy with representative biopsies taken from both normal and affected mucosa. RESULTS: After 1 yr there were macroscopical signs of progression in 14%; 22% showed regression, and 30% had a normal colonoscopy. The histological changes from diagnosis until follow-up showed progression in 20%, 24% showed regression, and 24% had normal histological findings. Histological examination showed more extensive disease than did direct visualization in 4% of patients at diagnosis and in 28% at follow-up, whereas direct visualization showed more extensive disease than did histological examination in 18% of patients at diagnosis and 12% at follow-up. The best correlation at both diagnosis and follow-up was seen in pancolitis (99% and 88%, respectively). CONCLUSIONS: With regard to the extent of colonic involvement in the UC patients, we found less agreement between endoscopic and histological evaluation at the follow-up examination than at the start of the study. This could indicate that biopsies represent a better indicator than endoscopical examination for long term prognosis. Further studies are needed to confirm this finding.

Antiproliferative effect of the tripeptide pyroGlu-Phe-GlyNH2 on murine melanocytes: transitory delay of cell growth in vitro and the cell cycle specificity.

Cell growth and differentiation in melanocyte cell populations are regulated by a wide range of bioactive substances. Recently, the tripeptide pyroGlu-Phe-GlyNH2 which inhibits melanocyte growth in vitro was identified in both murine nontransformed melanocytes and malignant melanoma cells. The present study was undertaken to investigate the cell cycle specificity as well as the growth inhibitory profile of the tripeptide after a single or repeated administration to melanocyte cultures. Dose-related effects of the peptide were studied using three different bioassay systems: estimation of cell number, DNA synthesis, and cell flux into mitosis. Growth of melanocyte cultures as well as melanocyte mitotic activity were found to be reduced significantly by the tripeptide at two separate dose levels (10(-11) and 10(-14)-10(-15) M). Growth inhibition of melanocyte population did not last long: less than 36 h after the first and less than 24 h after the second peptide addition to the cultures. The level of DNA synthesis in melanocytes remained unchanged after a single peptide administration. The findings indicate that the tripeptide pyroGlu-Phe-GlyNH2 causes transitory delay of cell growth in cultured melanocyte population resulting from a reversible inhibition of melanocyte transition from the G2-phase of the cell cycle into mitosis.

Diagnostic utility of the polymerase chain reaction in 2 cases of suspected Whipple disease.

We describe 2 patients with a diagnosis of Whipple disease in whom the usual antibiotic therapy failed. A polymerase chain reaction-based test was used to identify the recently described Whipple bacillus, Tropheryma whippelii. In one case, the diagnosis was confirmed, whereas in the second case, which had been histologically diagnosed as Whipple disease of the brain, the process was identified as a monocyte-derived histiocytosis. In conclusion, Whipple disease can be distinguished from other diseases with similar histological features with the use of a polymerase chain reaction-based test.

Identification of a melanocyte growth-inhibiting tripeptide and determination of its structure.

The function and proliferation of melanocyte cell populations are influenced by a wide range of hormones and growth factors. Local cell renewal after sudden melanocyte loss appears to be regulated according to a negative feedback principle, however. In accordance with this assumption, we have examined growth-inhibitory activity in water extracts of cultured non-transformed melanocytes and melanoma cells (B16 cells). The extracts were fractionated by gel filtration on Sephadex G-25 and Fractogel MG 2000, by ion-exchange chromatography on DEAE-cellulose and Dowex 50 and by reverse-phase high-performance liquid chromatography on Bondesil and Partisil columns. Two peptides were isolated with the structures pyroGlu-Phe-GlyNH2 and pyroGlu-Cys-GlyNH2 as revealed by mass spectrometry, peptide sequencing and amino acid analysis. The two peptides were synthesized and tested for the ability to inhibit the growth of melanocyte cultures. Only pyroGlu-Phe-GlyNH2 was inhibitory. The dose-response curve was bell-shaped with maximum inhibition around 10(-15) M. The melanocyte tripeptide thus appears to be a new member of a group of N-substituted growth-regulating oligopeptides found in other tissues.

Interferon-alpha 2b therapy in low-activity hepatitis C: a pilot study.

BACKGROUND: Many patients with chronic hepatitis C have long periods of normal or near-normal liver enzyme levels, even though histologic alterations have been confirmed. The recommendation today is not to treat this patient group. METHODS: In a pilot study 23 hepatitis C virus (HCV) RNA-positive patients with alanine aminotransferase (ALAT) levels less than 1.5 times upper normal limits for at least 6 months on more than three occasions and with histologic liver abnormalities compatible with chronic hepatitis C were treated with 3 MU of interferon-alpha 2b three times a week for 6 months. RESULTS: Nine patients (39%) became HCV RNA-negative in serum during treatment, but only two (8.7%) remained so after 6 months' follow-up. Significantly more patients with genotype other than type 1 became HCV RNA-negative than patients with genotype 1 during treatment (P = 0.005). CONCLUSIONS: Patients with low-activity chronic hepatitis C have a response to interferon-alpha treatment similar to that of patients with increased ALAT levels. Genotype seems to influence the rate of response.

Regeneration of rat corneal epithelium is delayed by the inhibitory epidermal pentapeptide (EPP).

The effect of the inhibitory epidermal pentapeptide (EPP) on regeneration of rat corneal epithelium was studied over a 24-h period after removal of the central part of the corneal epithelium by means of n-heptanol and scraping. Both unphosphorylated and phosphorylated EPP inhibited the mitotic rate and the formation of new cells to the same extent. Thus, the mitosis inhibitor that originally was isolated from mouse epidermis, acts even on the ectodermally derived corneal epithelial cells.

Monstrous ascites in hereditary haemorrhagic telangiectasia.

BACKGROUND: Hepatic involvement in hereditary haemorrhagic telangiectasia (HHT) consisting of fibrosis, telangiectases, and cirrhosis, has been reported as a relatively frequent finding. CASE: A 50-year-old man with HHT presented with monstrous ascites. Liver biopsy demonstrated multiple dilated sinusoids but not cirrhosis. There were no findings indicative of portal hypertension or malignant disease. Portal pressure, recorded in hepatic vein wedge position, was normal. Arteriography showed numerous hypervascular lesions throughout the liver. The clinical course has been stable for more than 2 years. CONCLUSION: No other reason for the monstrous ascites could be found. We thus hypothesize that this case of monstrous ascites is due to hepatic involvement in HHT, presenting as numerous vascular lesions throughout the liver.

Biologically active pyroglutamyl N-terminal oligopeptides: parts of larger molecules?

In 1984 we identified and characterized a growth-inhibiting pentapeptide, pyroGlu-Glu-Asp-Ser-GlyOH, [EPP] from mouse epidermis. Later, other pyroGlu N-terminal oligopeptides have been isolated and characterized from liver and mouse intestine. The three pyroGlu-terminal mitosis inhibitory peptides are structurally similar and have several biological properties in common. A number of questions remain, however, to be answered, e.g., a) Are the peptides part of larger molecules; b) Do they bind to specific receptors on the target cells; c) How are they related to other growth-modulating factors, and c) Are they coded for by genes that are related to known growth regulating proto-oncogenes, especially to growth suppressing genes. To search for soluble parts of possible receptors, or carrier molecules, in water extracts of mouse epidermis we have used affinity columns coated with EPP with either the N-terminal end or the carboxy-end free. Both types of column bind a 70 kD protein. The protein bound to the column with a free N-terminal end splits into two small components under reducing conditions. To look for larger molecules of which EPP could be a fragment, we have used western blotting techniques and a polyclonal rabbit antiserum against EPP. Preliminary experiments have indicated that two different molecules bind to the antiserum.

Zinc and the mitosis-inhibitory epidermal pentapeptide (EPP) form a stimulatory chelated dimer.

A single intraperitoneal (i.p.) injection of picomolar doses of the epidermal pentapeptide (EPP), pGlu-Glu-Asp-Ser-GlyOH, is followed by a reversible inhibition of mouse epidermal cell proliferation. An equimolar mixture of zinc and EPP injected i.p. into hairless mice reversed the inhibitory activity, resulting in an immediate stimulation of epidermal G2-M cell flux. The stimulatory effect was strongest at the lowest dose (5 pmol). This effect was probably caused by a dizinc-dipentapeptide dimer, as shown by gel filtration and atomic emission spectrometry. When zinc was added in excess (EPP:Zn 1:9) no such dimer could be identified, and the mixture had no stimulatory effect. The results are discussed in terms of epidermal cell kinetics, and in relation to the use of zinc in dermatology.

Risk factors in primary sclerosing cholangitis.

The increasing use of liver transplantation and new treatment regimens requires an accurate estimate of the prognosis in primary sclerosing cholangitis. To clarify the natural history and prognosis of this disease, we studied the clinical features at the time of presentation and the outcome in 77 consecutive patients admitted to our hospital. The median age at diagnosis of primary sclerosing cholangitis was 32.5 years; 66% of the patients were male; 76 had concomitant inflammatory bowel disease and two had celiac disease. Thirty-four patients were classified as asymptomatic at diagnosis of primary sclerosing cholangitis. The mean follow-up time was 6.2 years; 25 patients have died or been transplanted. Cholangiocarcinoma has been diagnosed in 11 patients (14%). Female patients have a significantly poorer survival rate than male patients. The bilirubin level was found to be an independent risk factor for both mortality/transplantation, and for the occurrence of cholangiocarcinoma. Age at diagnosis of primary sclerosing cholangitis was an additional risk factor of death/transplantation. As bilirubin is an important prognostic factor for the development of both cholangiocarcinoma and death/transplantation, the construction of prognostic indices seems to be of limited value in the timing of transplantation of the individual patient.

Hepatobiliary disease in ulcerative colitis. An analysis of 18 patients with hepatobiliary lesions classified as small-duct primary sclerosing cholangitis.

BACKGROUND: The aim of the present study was to describe the characteristics of patients with ulcerative colitis (UC) and hepatobiliary disease that does not satisfy the diagnostic cholangiographic criteria of primary sclerosing cholangitis (PSC) and to compare this group with PSC patients. METHODS: Among 199 patients with UC admitted to our department during 1986-91, 64 patients had major hepatobiliary disease considered to be associated with the colitis. Biochemical tests, colonoscopy, endoscopic retrograde cholangiography (ERC), and liver biopsy were performed in these 64 patients and in 5 patients from our outpatient clinic. RESULTS: PSC was diagnosed in 51 patients (group I; 80%). The other 13 patients (20%) and the additional 5 patients (n = 18; group II) all had normal extrahepatic bile ducts. Five patients in group II also had normal intrahepatic ducts, whereas 13 patients had intrahepatic abnormalities. The male to female ratio in group II was 2.0:1. All of them had extensive colitis. The clinical symptoms and the biochemical and histologic findings were quite similar in groups I and II. CONCLUSIONS: The patients in group II of this study constitute a major group with hepatobiliary lesions associated with UC, amounting to one-fourth the number of PSC patients. They have several similarities with classical PSC of the large bile ducts, and we suggest that they be classified as having small-duct PSC.

An intraocular paraganglioma?

A tumour of the iris with the morphological and immunohistochemical characteristics of a paraganglioma is presented. Neither this type of tumour, nor the specialized nerve cells giving rise to the tumour, have previously been reported in the eye. A successful local resection was performed. The diagnosis and concept of paragangliomas are discussed briefly.

Beta-receptor blockade by propranolol modifies the effect of the inhibitory, endogenous epidermal pentapeptide on epidermal cell flux at the G2-M transition but not at the G1-S transition.

The mitosis inhibitory pentapeptide, pGlu-Glu-Asp-Ser-GlyOH (EPP), which was isolated from mouse epidermis extracts, belongs to a group of growth inhibitory peptides that all have pyroglutamyl at the N-terminal end. Earlier experiments with crude or partially purified skin extracts have shown that the inhibitory effect could be enhanced by beta-receptor agonists and by dibutyryl cAMP, and that beta-receptor blockade could neutralise it. We now show that treatment with the beta receptor blocker propranolol before or after EPP treatment of hairless mice significantly modifies the effect of EPP on mouse epidermal cell proliferation, as estimated by using a metaphase-arrest technique (Colcemid) to estimate the G2-M cell flux. The interaction between propranolol and EPP is complex; only the EPP-induced inhibition of the G2-M cell flux was modified by beta-receptor blockade, while the late (18-21 h) inhibition of the mitotic rate was unaltered. Propranolol alone was followed by a dose-related and transient increase in the epidermal mitotic rate. The phosphodiesterase inhibitor caffeine had no effect on its own on epidermal cell proliferation but counter-acted the late (18-21 h) EPP-induced inhibition.