Long-term evaluation of a rat model of chronic cholangitis resembling human primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic disorder with a presumed autoimmune aetiopathogenesis. We have recently described a novel organ-specific rat model of fibrosing cholangitis induced by intrabiliary administration of the hapten-reagent 2,4,6-trinitrobenzenesulfonic acid (TNBS) with similarities to human PSC. In the present report, we have evaluated the long-term outcome of TNBS-induced cholangitis in this model. Mild stenosis of the common bile duct of female Lewis rats (n = 18) was achieved by subtotal ligation and cholangitis induced by TNBS injection (50 mg/kg) into the dilated bile duct after a second laparotomy. After 8 and 12 months, we found no evidence of cholangitis in serum chemistry or histology or retrograde cholangiography of TNBS-treated rats. Antineutrophil cytoplasmic antibodies were positive in 75% of animals but were not predictive of liver damage. Tumour necrosis factor-alpha levels were not elevated in serum or in mononuclear spleen cell supernatants. Our findings suggest that a single initial insult is not sufficient to trigger chronic progressive inflammation. Rather, perpetuation of inflammation probably requires additional stimuli.

Noninvasive assessment of Crohn's disease activity: a comparison of 18F-fluorodeoxyglucose positron emission tomography, hydromagnetic resonance imaging, and granulocyte scintigraphy with labeled antibodies.

OBJECTIVES: Detection of disease activity in Crohn's disease (CD) is of crucial importance for diagnosis and management of the disease. Noninvasive methods for monitoring are desirable and comprise hydromagnetic resonance imaging (hydro-MRI) and leukocyte scintigraphy. In addition, a recent case report indicated the potential of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) to assess CD activity. However, comparative prospective studies are lacking. METHODS: Between February, 1999 and August, 2000, 59 patients with CD were enrolled in a prospective study to assess disease activity by FDG-PET, hydro-MRI, and immunoscintigraphy with anti-nonspecific cross-reacting antigen 95 antigranulocyte antibodies. In 28 of these patients, colonoscopy could be performed. Twelve patients with irritable bowel syndrome and 20 tumor patients without gut inflammation served as controls. Results were compared by statistical analysis. RESULTS: FDG-PET detected 127 pathological findings (average maximum standardized uptake value = 4.4 +/- 1.1) in the terminal/neoterminal ileum (37), small bowel (24), and colon (66) of 54 patients with CD, whereas no pathological findings were seen in five patients with CD, the control patients with irritable bowel syndrome, and the tumor patients without gut inflammation. In contrast, examination with hydro-MRI or granulocyte antibodies detected less pathological findings in CD patients. Forty-five of the detected foci were accessible to endoscopic verification. The correlation of the foci with endoscopic findings showed a high specificity (>89%) of all three methods to detect inflamed areas in the terminal ileum and colon of patients with CD, although analyses by hydro-MRI and granulocyte antibody scan had strikingly lower sensitivities (40.9% and 66.7%) than FDG-PET analysis (85.4%). CONCLUSIONS: FDG-PET appears to be a reliable noninvasive tool for simultaneous detection of inflamed areas in the small and large bowel of patients with CD. FDG-PET can be used to detect disease activity in the terminal ileum and colon of CD patients with high sensitivity and specificity.

[Hydro-MRI in inflammatory bowel diseases: a comparison with colonoscopy and histology]. / Hydro-MRT bei entzündlichen Darmerkrankungen--Eine koloskopisch-histologische Vergleichsstudie.

PURPOSE: To compare hydro-MRI with colonoscopy and biopsy specimen regarding the assessment of inflammatory activity and the differentiation of inflammatory bowel diseases. MATERIAL AND METHODS: After an oral bowel opacification using 1000 ml of a 2.5% mannitol solution and a rectal bowel opacification using 250-500 ml of a 0.9% saline solution, axial and coronal breath-hold sequences +/- Gd-DTPA (HASTE-["half-Fourier acquisition single-shot turbo spin echo"] and dynamic FLASH-["fast low angle shot"]) were acquired in 27 patients with inflammatory bowel disease. The enhancement of the bowel wall as well as morphological MRI findings were correlated with colonoscopy and biopsy specimens. By means of the MRI findings, Crohn's disease (CD) and ulcerative colitis (UC) should be differentiated. RESULTS: In CD, a significant correlation between the contrast enhancement of the inflamed bowel wall (delta SI) and the endoscopic/histopathologic indices could be established (r = 0.52; p = 0.02 and r = 0.72; p = 0.001). In UC, no correlations between delta SI and the endoscopic/histopathologic indices could be found. The correct diagnosis of CD and UC by MRI findings was possible in 22/27 patients (81%). CONCLUSION: Hydro-MRI with dynamic studies is suitable for the assessment of disease activity in CD, but unreliable in UC. Hydro-MRI provides useful information for the differentiation of CD and UC.

A novel rat model of chronic fibrosing cholangitis induced by local administration of a hapten reagent into the dilated bile duct is associated with increased TNF-alpha production and autoantibodies.

BACKGROUND/AIM: The cholangiopathies represent hepatobiliary diseases in which bile-duct epithelial cells are targets for destructive processes, including immune-mediated damage. We describe a novel rat model of chronic fibrosing cholangitis induced by administration of the hapten reagent 2,4,6-trinitrobenzenesulfonic acid (TNBS) into the dilated bile duct. METHODS: The common bile duct was dilated due to a mild stenosis in 8-week-old female Lewis rats. TNBS (50 mg/kg) was injected during a second laparotomy. RESULTS: TNBS-treatment reproducibly resulted in chronic fibrosing cholangitis. In retrograde cholangiography the bile ducts showed irregularities, beading and strictures. Alkaline phosphatase levels remained abnormal throughout the study period. Immunohistochemical staining showed an increased number of macrophages, CD3+ T-lympbocytes and MHC class II antigen upregulation. The spontaneous interferon-gamma, tumor necrosis factor-alpha and interleukin-10 production of liver-derived mononuclear cells was increased. Anti-neutrophil cytoplasmic antibodies with specificity against myeloperoxidase, catalase and actin were found between 1 and 12 weeks after TNBS injection. CONCLUSIONS: We established a novel rat model of chronic fibrosing cholangitis with histologic, cholangiographic, serologic and immunologic similarities to human primary sclerosing cholangitis. This model may be used to study pathomechanisms of chronic cholangitis without concomitant inflammatory bowel disease.

Hydro-MRI in Crohn's disease: appraisal of disease activity.

RATIONALE AND OBJECTIVES: To appraise the value of hydro-MRI in the assessment of activity in Crohn's disease. METHODS: After bowel opacification with 1000 mL of an orally administered 2.5% mannitol solution was achieved, axial and coronal breath-hold sequences (T2-weighted half-Fourier acquisition single-shot turbo spin-echo sequences with or without fat saturation, dynamic T1-weighted fast low-angle shot sequences, and contrast-enhanced T1-weighted fast low-angle shot with fat saturation sequences) were acquired in 82 patients with proved Crohn's disease at 1.0 T. Enhancement of the bowel wall was correlated with other MRI findings, with the Crohn's disease activity index (CDAI), and with levels of C-reactive protein (CRP). RESULTS: In Crohn's disease, contrast enhancement of the affected bowel wall was markedly increased in comparison with the normal bowel wall (+80% +/- 22% versus +43% +/- 12%; P = 3 x 10(-15)). Positive correlations could be established between the increase in bowel wall enhancement and many other MRI findings. Between the increase in bowel wall enhancement and the CDAI, only a poor correlation was found (r = 0.25, P = 0.02). There was no statistical correlation between the increase in bowel wall enhancement and CRP. CONCLUSIONS: Hydro-MRI seems to be superior to the CDAI and CRP for the registration of Crohn's disease activity. In particular, differentiation between an active and an inactive (scarred) stenosis, which is crucial for the choice of therapeutic procedures, seems to be more reliable by the interpretation of several morphological and functional parameters on hydro-MRI than by the use of CDAI and CRP.

[Assessment of inflammatory activity in Crohn disease with hydro-MRI]. / Beurteilung der entzündlichen Aktivität des M. Crohn mit der Hydro-MRT.

PURPOSE: To assess the value of hydro-MRI in the assessment of the activity of Crohn's disease. MATERIALS AND METHODS: After an oral bowel opacification using 1000 ml of a 2.5% mannitol solution, axial and coronal breath-hold sequences (T2W HASTE +/- FS, contrast-enhanced T1W FLASH FS) were acquired in 63 patients with Crohn's disease at 1.0 T. The enhancement of the bowel wall was correlated with other MRI findings, with the Crohn's disease activity index (CDAI), and the C-reactive protein (CRP). RESULTS: In Crohn's disease, contrast enhancement of the affected bowel wall is markedly increased in comparison with the normal bowel wall (+80 +/- 23% vs. +43 +/- 11%; p = 8 x 10(-11)). Positive correlations could be established between the increase of bowel wall enhancement and other MRI findings. Between the increase of bowel wall enhancement and the CDAI a poor correlation was found (r = 0.25; p = 0.046). There was no statistical correlation between the increase of bowel wall enhancement and the CRP (r = 0.09; p = 0.24). CONCLUSION: Hydro-MRI allows an assessment of the activity of Crohn's disease.

Anti-neutrophil cytoplasmic antibodies in a rat model of trinitrobenzenesulphonic acid-induced liver injury.

BACKGROUND: In sera from patients with autoimmune liver diseases, e. g. primary sclerosing cholangitis (PSC) and autoimmune hepatitis, anti-neutrophil cytoplasmic antibodies (ANCAs) can be found. Until now, no animal model of ANCA induction in liver disease has been described. In this study, we describe a novel rat model of acute liver injury and subsequent ANCA production. MATERIALS AND METHODS: The hapten reagent 2,4,6-trinitrobenzenesulphonic acid (TNBS) was injected into the portal vein of female Lewis rats. Two experimental groups were studied: group A (TNBS/ethanol) received different TNBS concentrations; control animals of group B (ethanol) were injected with 10% (v/v) ethanol/0.9% (w/v) NaCl. RESULTS: A dose-dependent acute necrotizing liver injury occurred after injection of TNBS. Histopathological examination revealed acute hepatic injury with confluent parenchymal necrosis, mild bile duct proliferation and periportal infiltration. The periportal infiltration consisted mainly of macrophages and T lymphocytes. ANCAs were found in an allogenic test system between 1 and 8 weeks after TNBS injection in 11 out of 28 (39%) TNBS-treated rats (group A) and did not correlate with the extent of liver injury or TNBS dose. Autoantibody specificities of IgG type were directed against catalase (29%), myeloperoxidase (14%) and actin (7%), as detected by enzyme-linked immunosorbent assay and Western blotting. Moreover, autoantibodies against the asialoglycoprotein receptor were observed. Peripheral blood mononuclear cells and spleen lymphocytes from TNBS-treated rats were shown to produce ANCAs. CONCLUSION: In summary, we were able to show that intraportal administration of the hapten reagent TNBS induces an acute necrotizing liver injury with subsequent ANCA production in Lewis rats. ANCA specificities were mainly directed against catalase, an autoantigen that has recently been identified in sera from patients with primary sclerosing cholangitis and autoimmune hepatitis. This animal model offers the opportunity to study the pathomechanisms of ANCA production in necrotizing liver injury.

[Hydro-MRT with fast sequences in Crohn's disease: a comparison with fractionated gastrointestinal passage]. / Hydro-MRT mit schnellen Sequenzen bei Morbus Crohn: Vergleich mit der fraktionierten Magen-Darm-Passage.

PURPOSE: To compare the value of hydro-MRI with that of barium studies in patients with Crohn's disease. MATERIALS AND METHODS: After an oral bowel opacification using 1000 ml of a 2.5% mannitol solution, axial and coronal breath-hold sequences (T2W HASTE +/- FS, contrast-enhanced T1W FLASH FS) were acquired in 46 patients with Crohn's disease at 1.0 T. The findings of hydro-MRI were compared with those of barium studies. RESULTS: In the stomach and the small bowel, hydro-MRI and barium studies demonstrated similar numbers of Crohn's involvements (39 vs. 36); in the colon, hydro-MRI showed clearly more affections (23 vs. 10). Hydro-MRI showed 12.7 cm of inflamed bowel per patient, on average (barium studies: 10.4 cm; p = 0.004). There was a good agreement between the two methods regarding the assessment of the extent of Crohn's disease and the severity of bowel stenoses (r = 0.89 and 0.88, respectively). CONCLUSIONS: For the assessment of Crohn's disease, hydro-MRI is preferable to the barium study because of the superior imaging quality and the lack of radiation exposure.

Signal transduction pathways of membrane expression of proteinase 3 (PR-3) in human endothelial cells.

At present, the exact mechanism of the pathogenic effect of anti-PR-3 antibodies remains unknown. Interaction of anti-neutrophil cytoplasmic antibodies (ANCAs) with human umbilical vein endothelial cells (HUVECs) may play a key role. Recently we were able to show that ANCAs recognize their target antigen, PR-3, translocated into the membrane of HUVECs. The objective of this study was to investigate regulation, i.e. signal transduction pathways, of PR-3 expression in endothelial cells. HUVECs were isolated according to the method of Jaffe et al. and cultured under standard conditions. A cyto-enzyme-linked immunosorbent assay (ELISA) with unfixed cells was performed. Membrane-expressed PR-3 was detected by affinity-purified and monoclonal anti-PR-3 Ab. Tumour necrosis factor alpha (TNF-alpha)-induced membrane expression of PR-3 could be blocked with the RNA synthesis inhibitor actinomycin D, the protein kinase C (PKC) and proteinase A (PKA) inhibitor staurosporine, the specific PKA inhibitor calphostin C, the c-AMP-dependent PKA inhibitor KT5720 and the tyrosine kinase inhibitor genistein in a dose-dependent manner. The effect of calphostin C was the most significant. In addition, the effect of phorbol 12-myristate 13-acetate (PMA), a mediator of intracellular second messengers, was investigated. In our study, pretreatment of cells with PMA for 48 h led to a down-regulation of PR-3 expression. This effect, however, could be overridden by TNF-alpha stimulation, i.e. TNF-alpha-induced membrane expression of PR-3 was resistant to down-regulation of PKC. In conclusion, our data suggest that translocation of PR-3 in HUVECs is an active process depending on protein synthesis. PR-3 expression by HUVECs may involve a PKC reactive to cytokines such as TNF-alpha which induces PR-3 expression at a transcriptional level.

[Prostate carcinoma associated spontaneous factor VIII:C inhibitor hemophilia. Successful therapy of severe hemorrhagic complication with porcine factor VIII in a 75-year-old patient]. / Prostatakarzinom-assoziierte spontane Hemmkörperhämophilia. Erfolgreiche Therapie einer schweren Blutungskomplikation mit porzinem Faktor VIII bei einem 75jährigen Patienten.

Inhibitors of factor VIII are a rare condition in non-hemophiliacs, but they are frequently responsible for life threatening hemorrhage. Acquired factor VIII:C inhibitors represent the spontaneous development of autoantibodies that partially or completely neutralize the plasma coagulant activity of the clotting factor. The autoantibodies can arise in diverse clinical settings, in older adults they are frequently associated with immunologic disorders or malignancies. We report of a 75-year-old man with acquired factor VIII:C inhibitor associated with adenocarcinoma of the prostate and a successful treatment of a severe bleeding complication with porcine factor VIII. A 75-year-old man was admitted because of a hematoma of his right cheek and an isolated prolonged aPTT. Acquired factor VIII:C inhibitor was identified as the cause and immuno-suppressive therapy was begun. In the clinical course severe hemorrhaging occurred and was successfully treated with porcine factor VIII (Hyate:C). The initially high inhibitor titer of 32 Bethesda Units (BU) disappeared. As the cause of acquired factor VIII:C inhibitor a newly diagnosed adenocarcinoma of the prostate is likely. After complete remission of acquired factor VIII:C inhibitor radiation therapy was begun. Six months after severe hemorrhaging the patient was clinically stable and PSA levels were normal. This case demonstrates the necessity of a precise diagnosis and therapy regimen of this coagulopathy based on clinical and laboratory data. In the absence of hemorrhage immuno-suppressive therapy with corticosteroids is indicated, in a patient with severe bleeding and high inhibitor titer (> or = 5 BU) porcine factor VIII should be administered.

Characterization of target antigens from anti-neutrophil cytoplasmic antibodies in autoimmune hepatitis type-I.

The occurrence of anti-neutrophil cytoplasmic antibodies (ANCA) has been described in sera of patients with autoimmune hepatitis (AIH). The significance of this finding remains uncertain and the nature of the target antigen(s) has not yet been defined. We studied 32 sera from patients with AIH type-I and prepared extracts of human neutrophils to identify the target antigen(s). A 43 kDa dominant immunoreactive protein was found and identified as the cytoskeletal component actin. Initial studies to define the antigenic determinants identified three different actin domains.

Antineutrophil nuclear antibodies (ANNA) in primary biliary cirrhosis: their prevalence and antigen specificity.

Antineutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies first associated with Wegener's granulomatosis (WG). In autoimmune liver diseases, ANCA have been described recently in patients with primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH) and primary sclerosing cholangitis (PSC). Controversy exists about the prevalence and specificity of ANCA in patients with autoimmune liver diseases. The purpose of this study was first to assess the prevalence of antineutrophil antibodies in patients with primary biliary cirrhosis and second to identify possible target antigens of antineutrophil antibodies. Sera from 33 patients with PBC, 75 patients with AIH, 16 patients with PSC, 90 control sera (chronic hepatitis B, chronic hepatitis C, alcoholic liver cirrhosis, systemic lupus erythematosus) and sera from healthy blood donors were enrolled in the present study. ANA and ANCA were detected using standard protocols, antibodies against the nuclear antigen SP100 were detected by ELISA with recombinant antigen, antibodies against neutrophil alpha-granules were detected by ELISA. P-ANCA were found in two of 33 PBC sera, both patients presented with PBC/AIH overlap syndrome. In six of 33 (18%) PBC sera a nuclear dot like immunofluorescence pattern (antineutrophil nuclear antibodies, ANNA) was observed. These dots were small in size and located all over the nucleus, the anti-SP100 fluorescence. This result was confirmed by IFT on HEp-2 cells and by ELISA. High tier antibodies against SP100 were found to be specific for patients with PBC. Comparing the IFT results on HEp-2 cells and human neutrophils, we found an excellent correlation (Spearman's rank correlation coefficient = 0.968, P < 0.001). ANCA were detected in AIH type-1 (75%), SLA-positive AIH (36%) and PSC (63%). AIH type-2 was found to be ANCA negative. All PSC contained no antibodies against neutrophil alpha-granules. Our results show: 1. P-ANCA in patients with PBC indicate a PBC/AIH overlap syndrome; 2. ANNA in patients with PBC are mostly directed against SP100; 3. neutrophil granule components are not the ANCA specific antigens in patients with PSC.

Actin is a target antigen of anti-neutrophil cytoplasmic antibodies (ANCA) in autoimmune hepatitis type-1.

BACKGROUND/AIM: Anti-neutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies first associated with Wegener's granulomatosis and microscopic polyangiitis. The significance of ANCA in autoimmune hepatitis remains uncertain; the nature of the antigen or antigens has not been defined yet. The purpose of this study was to identify the target antigen of ANCA in patients with autoimmune hepatitis. METHODS/RESULTS: Sera from 32 type-1 autoimmune hepatitis patients were used in the present study. ANCA were detected in 24 of 32 sera (75%). A diffuse cytoplasmic staining pattern (C-ANCA) was detected in 14 patients; the P-ANCA pattern was observed in 10 patients. An extract of human neutrophils was prepared and subjected to SDS-PAGE and Western Blot analysis. A 43-kD dominant immunoreactive protein was found in 20 (63%) autoimmune hepatitis patients. Aminoacid sequence analysis of the 43 kD protein identified actin. Cytoplasmic or perinuclear staining pattern could be reduced after absorption of sera with actin and after removing anti-actin antibodies by affinity chromatography. This was observed for all C-ANCA and for 8 out of 10 P-ANCA. Moreover in double-staining indirect immunofluorescence, the same type of diffuse cytoplasmic staining was observed with autoimmune hepatitis-sera and anti-actin antibodies. In Western Blot analysis with actin, 17 (53%) patients gave a positive result, while 15 (47%) patients had a positive actin-ELISA. CONCLUSION: This is the first report to identify the cytoskeletal protein actin as an ANCA antigen.

Antibodies to proteinase 3 mediate expression of vascular cell adhesion molecule-1 (VCAM-1).

VCAM-1 was first identified as an adhesion molecule induced on human endothelial cells (HEC) by inflammatory cytokines such as IL-1, tumour necrosis factor (TNF), and lipopolysaccharide (LPS). The molecule binds to a variety of leucocytes, including B cells, T cells, basophils, eosinophils and monocytes. Vascular expression of VCAM-1 has been associated with a number of disease states, including rheumatoid arthritis and vasculitis. The detection of anti-neutrophil cytoplasmic antibodies (ANCA), especially to proteinase 3 (PR3), has become important in the diagnosis of Wegener's granulomatosis (WG) and related vasculitides. Recently we were able to demonstrate a direct effect of anti-PR3 antibodies on neutrophil-endothelial interactions (Blood 1993; 82:1221). Binding of anti-PR3 antibodies to their antigen translocated into the membrane of HEC leads to an enhanced adhesion of neutrophils via induction of E-selectin (Clin Exp Immunol 1993; 94:440). The aim of this study was to investigate the effect of anti-PR3 antibodies on the expression of VCAM-1. HEC were isolated from umbilical vein and cultured on microtitre plates. After preincubation with purified anti-PR3 antibody, purified control antibodies (SS-A, SS-B, RNP) (IgG and F(ab')2 fragments) or different cytokines (controls), VCAM-1 was detected on the surface of unfixed HEC by cyto-ELISA and polymerase chain reaction analysis. Incubation of HEC with anti-PR3 antibodies led to a marked increase of endothelial VCAM-1 expression with a peak after 8 h. Incubation with TNF-alpha also led to maximal VCAM-1 expression after 4-6 h (control). Increased adhesion of T lymphocytes to HEC after binding of anti-PR3 antibodies to their antigen could be confirmed by performing adherence assays. This effect could be inhibited by antibodies to VLA-4. In conclusion, we have been able to show that cytokine-like effects of anti-PR3 antibodies on HEC are not limited to induction of neutrophil adhesion. Anti-PR3 antibodies may thus contribute to the regulation of T lymphocyte migration from the blood by HEC in ANCA-related vasculitides.

[Long-term damage to duodenal mucosa in malabsorption syndrome as a sequela of Giardia lamblia infection]. / Langanhaltende Schädigung der Duodenalschleimhaut mit Malabsorptionssyndrom als Folge einer Infektion mit Giardia lamblia.

A 35-year-old German woman presented after a three month journey to Sudan with intermittent, persistent watery diarrhea. Stool examination identified Giardia lamblia. The patient was unsuccessfully orally treated with metronidazol 250 mg three times a day for seven days and tinidazol 2 g as single dose. During a two week hospitalization we observed a total villous atrophy in a duodenal biopsy specimen in absence of celiac sprue or a immunodeficiency syndrome. We treated with metronidazol 500 mg three times a day intravenously and discharged the patient in good condition. This case report shows the variability of clinical presentation of infection with Giardia lamblia. Moreover, we demonstrate rarely found histological changes as reason for clinical presentation, unsuccessful oral treatment and malabsorption. The intravenous therapy lead to a permanent eradication of Giardia lamblia.

[Reversible myelofibrosis in angioimmunoblastic lymphadenopathy]. / Reversible myelofibrose bei angioimmunoblastischer Lymphadenopathie.

Ankle oedema and abdominal swelling suddenly developed in a 55-year-old woman who also had lymphadenopathy in the neck, axillae and groin. Ultrasonography demonstrated hepatosplenomegaly, ascites and pleural effusions. Histological examination of some lymph-nodes from the axilla and groin revealed angioimmunoblastic lymphadenopathy (low-malignant peripheral T cell lymphoma). Bone-marrow biopsy was undertaken because of a normocytic anaemia (haemoglobin 4.9 g/dl) requiring blood transfusion, thrombocytopenia (5000/microliters) and monoclonal IgG gammopathy. This showed lymphoma-associated secondary myelofibrosis. Treatment with prednisone (2 mg/kg daily for 8 weeks) and vincristine (1 mg/m2 once weekly for 4 weeks) brought about partial remission of the angioimmunoblastic lymphadenopathy with normalization of the clinical and laboratory findings, the splenohepatomegaly regressed, and there was only a small amount of ascites. Four months after onset of the illness bone-marrow biopsy also showed regression of the myelofibrosis.