[Show abstract][Hide abstract] ABSTRACT: Blood group-related glycans determining ABO and Lewis blood groups are known to function as attachment factors for most of the norovirus (NoV) strains. To identify binding specificity of each NoV, recombinant norovirus-like particles (VLPs) and human saliva samples with different ABO, Lewis phenotypes and secretor status have been commonly applied. When binding specificities of VLPs prepared from 16 different genotypes of NoVs in GI and GII genogroups were characterized in samples of human gastric mucosa compared to human saliva based on blood group phenotypes, considerable differences were observed for several strains. Novel binding specificities determined by an ELISA using preparations from human gastric mucosa were also ascertained by immunohistochemical analyses using human jejunal mucosa, widely believed to be susceptible to NoV infection. Further, A, B and O(H) blood group substances prepared from porcine and squid tissues were found to be effective for preventing ABO blood group-specific binding of VLPs to both saliva and mucosa samples. Therefore, these blood group substances might have potential for the prevention and treatment of NoV infection.
[Show abstract][Hide abstract] ABSTRACT: Human plasma
-acid glycoprotein (AGP) from cancer patients and healthy volunteers was purified by sequential application of ion-exchange columns, and N-linked glycans enzymatically released from AGP were labeled and applied to a mass spectrometer. Additionally, a novel software system for use in combination with a mass spectrometer to determine N-linked glycans in AGP was developed. A database with 607 glycans including 453 different glycan structures that were theoretically predicted to be present in AGP was prepared for designing the software called AGPAS. This AGPAS was applied to determine relative abundance of each glycan in the AGP molecules based on mass spectra. It was found that the relative abundance of fucosylated glycans in tri- and tetra-antennary structures (FUCAGP) was significantly higher in cancer patients as compared with the healthy group (
). Furthermore, extremely elevated levels of FUCAGP were found specifically in patients with a poor prognosis but not in patients with a good prognosis. In conclusion, the present software system allowed rapid determination of the primary structures of AGP glycans. The fucosylated glycans as novel tumor markers have clinical relevance in the diagnosis and assessment of cancer progression as well as patient prognosis.
[Show abstract][Hide abstract] ABSTRACT: Cellulose acetate (CA) beads are often used for leucocyte apheresis therapy against inflammatory bowel disease. In order to clarify the mechanism of the anti-inflammatory effects of CA, global analysis of the molecules generated in blood by the interaction with CA beads was performed in this study. An activated medium was collected from whole blood that had been preincubated with CA beads, and the effects of the CA-activated medium on leucocyte function were investigated. Fresh blood was stimulated with lipopolysaccharide (LPS) or interferon (IFN)-β in the presence of the activated medium, and levels of chemokines and cytokines, including CXCL10 (IFN-inducible protein-10), and phosphorylated STAT1 (signal transducer and activator of transcription 1), which is known to be essential for CXCL10 production in leucocytes, were measured. IFN-β- or LPS-induced CXCL10 production, expression of CXCL10 mRNA and phosphorylation of STAT1 were significantly reduced in the presence of the medium pretreated with CA beads compared with the control without the CA bead treatment. The factors inhibiting CXCL10 production were identified as the C3 and C4 fragments by mass spectrometry. The monomeric C3bi and C4b proteins were abundant in the medium pretreated with CA beads. Furthermore, purified C3bi and C4b were found to inhibit IFN-β-induced CXCL10 production and STAT1 phosphorylation. Thus, STAT1-mediated CXCL10 production induced by stimulation with LPS or IFN was potently inhibited by monomeric C3bi and C4b generated by the interaction of blood with CA beads. These mechanisms mediated by monomeric C3bi and C4b may be involved in the anti-inflammatory effects of CA.
[Show abstract][Hide abstract] ABSTRACT: Diet is known to have a major role in the expression of inflammatory bowel disease (IBD). Thus, dietary monitoring, mostly in small cohorts of patients' report that intake of certain nutritional constituents like fat, refined sugar, fruits, vegetables and fibre impacted on the progression of IBD. These findings are often compromised by insufficient data or methodological limitations and do not provide realistic data on any given dietary factor. Among various dietary interventions or supplements, probiotics seem to offer some benefit, albeit meta-analyses indicated no significant efficacy. Enteral nutrition appears to be effective in both active and quiescent Crohn's disease (CD). Yet, independent meta-analyses have shown enteral nutrition to be inferior to corticosteroids in the management of active CD. In conclusion, our current knowledge on dietary risk factors for IBD and the therapeutic benefit of appropriate dietary interventions need to be strengthened by future studies in large cohorts of patients.
Full-text · Article · Jan 2011 · Practical gastroenterology
[Show abstract][Hide abstract] ABSTRACT: Patients with active inflammatory bowel disease (IBD) have elevated and activated myeloid leucocytes which infiltrate the colonic mucosa in vast numbers. Myeloid leucocytes such as the CD14(+) CD16(+) monocytes are major sources of tumour necrosis factor (TNF)-α, and therefore selective granulocyte/monocyte (GM) adsorption (GMA) should promote remission or enhance efficacy of pharmacological therapy. However, studies in IBD have reported both impressive as well as disappointing efficacy outcomes, indicating that patients' demographic factors might determine responders or non-responders to GMA. Nonetheless, this non-drug intervention has an excellent safety profile, and therapeutic GMA is expected to expand. In this review, attempts have been made to compile an update on the mode of actions (MoA) of the Adacolumn GMA. The MoA of GMA appears to be more than adsorption of excess neutrophils and TNF-producing CD14(+) CD16(+) monocytes per se. Adsorbed GMs release interleukin (IL)-1 receptor antagonist, hepatocyte growth factor and soluble TNF receptors, which are anti-inflammatory. Additionally, a sustained increase in lymphocytes including the regulatory CD4(+) CD25(+) T cells (lymphocyte sparing) is seen post-GMA. The impact of GMA on the immune system is potentially very interesting in the context of treating immune-related diseases. Future studies are expected to add intriguing insights to the MoA of GMA.
[Show abstract][Hide abstract] ABSTRACT: Diet is thought to have an important role in the immunopathogenesis and treatment of inflammatory bowel disease (IBD).
To identify dietary constituents as risk factors for development of IBD and the therapeutic efficacy of dietary modifications or enteral nutrition in IBD.
The Medline and the Cochrane Library were searched for clinical trials and meta-analyses in the scope of diet and nutrition in IBD.
There are many studies in small cohorts of patients that claim that intake of certain diet constituents like fat, refined sugar, fruits, vegetables and fibre affect the expression of IBD. These are often compromised by insufficient data or methodological limitations and do not provide unequivocal evidence to incriminate any particular dietary factor. Among various dietary interventions, none has shown striking efficacy with the possible exception of complete enteral nutrition. Enteral nutrition appears effective in both active and quiescent Crohn's disease (CD), but independent meta-analyses have shown enteral nutrition to be inferior to corticosteroids in the management of active CD, when assessed on an intention-to-treat basis.
The current levels of knowledge concerning dietary risk factors for IBD, and the therapeutic efficacy of dietary and nutritional interventions need to be supported by well-designed trials in large cohorts of patients.
[Show abstract][Hide abstract] ABSTRACT: Regulatory T cells (T(reg)) have an essential role in maintaining immune tolerance in the gut. The functional CD4(+) T(reg) express the transcription factor forkhead box protein 3 (FoxP3) or a CD25(high) in humans. Further, depletion of elevated granulocytes/monocytes by extracorporeal adsorption (GMA) induces immunomodulation in patients with ulcerative colitis (UC). We investigated the impact of GMA on T(reg). Thirty-one UC patients, clinical activity index (CAI) 12.1 +/- 2.97, refractory to conventional medications including intravenous corticosteroid and 13 healthy controls (HC), were included. Patients received five GMA sessions over 5 weeks. Biopsies from the rectal mucosa and blood samples at baseline and post-GMA were immunostained with anti-CD4/FoxP3 and anti-CD4/CD25 antibodies for immunohistochemistry and flow cytometry. Following GMA, 22 of 31 patients achieved remission (CAI <or= 4, P < 0.01) and their endoscopic activity index decreased from 10.6 +/- 2.32 to 4.75 +/- 1.48 (P = 0.003). The circulating CD4(+)CD25(high+) T(reg) level was low and increased markedly in responders (P < 0.02). In the nine non-responders, the baseline CD4(+)CD25(high+) T(reg) level was about 50% of the level in the responders (P < 0.03) or in the HC (P < 0.01), and all nine had to undergo colectomy. Conversely, the number of CD4(+)/FoxP3(+) mucosal T(reg) in GMA responders decreased significantly after the fifth GMA session compared with the baseline level (P < 0.05). It is believed that the CD4(+) T(reg) has an essential role in the control of immune pathology in UC patients and a net influx of these cells from the circulation into the mucosa may proceed to suppress inflammation. GMA can impact the circulating as well as the mucosal levels of T(reg).
[Show abstract][Hide abstract] ABSTRACT: The Adacolumn selectively depletes granulocytes and monocytes/macrophages, which are thought to be part of the immunopathogenesis of ulcerative colitis. This work aims at evaluating the safety and clinical efficacy of the Adacolumn in patients with ulcerative colitis in large population-based data sets.
The Adacolumn post marketing surveillance in Japan was undertaken on 697 patients in 53 medical institutions over 7 years from 29 October 1999 to 28 October 2006. Clinical efficacy and safety data were provided by patients' physicians in the participating institutes.
Safety was evaluated in all the 697 patients and efficacy in 656 patients. At entry, 92% of the patients were on salicylates, 74% on prednisolone and only 9% on immunomodulators. Approximately 40% of patients had severe ulcerative colitis and over 70% had ulcerative colitis that was refractory to conventional medications. There was no serious adverse events; mild to moderate adverse events were seen in 7.7% of the patients. The overall response (remission or significantly improved) was 77.3%; the remission rate based on clinical activity index was 71.1%, while 17.1% remained unchanged and 5.6% worsened. Patients were subgrouped into severe, moderate and mild ulcerative colitis based on clinical activity index (n=578), the response rates were 63.2%, 65.7% and 80.4%, respectively (P<0.001). Endoscopic assessment of efficacy showed very significant mucosal healing, Matts' endoscopic index improved from 3.2+/-0.04 to 2.1+/-0.7 (n=219, P<0.001); reduction in prednisolone dose (P<0.0001); leucocyte count (n=358, P<0.0001) and C-reactive protein (n=314, P<0.0001). Patients who received > or =6 Adacolumn sessions (n=319) did better than patients who received < or =5 sessions (n=188, P=0.004) and multivariate logistic regression analysis revealed that baseline granulocyte count was the strongest predictor of clinical response to Adacolumn (P=0.0191, odds ratio 1.151).
This post marketing surveillance provides the largest ever efficacy and safety data on the Adacolumn therapeutic leucocytapheresis in patients with ulcerative colitis. As a non-pharmacologic treatment for patients with active ulcerative colitis most of whom were refractory to conventional drug therapy, the observed efficacy was very significant. Baseline granulocyte count was convincingly an independent predictor of clinical response.
No preview · Article · Feb 2009 · Digestive and Liver Disease
[Show abstract][Hide abstract] ABSTRACT: Granulocyte, monocyte/macrophage adsorptive apheresis is a novel treatment for active ulcerative colitis. However, as yet no study has reported on a subset of patients who might respond well to granulocyte, monocyte/macrophage adsorptive apheresis therapy.
To identify factors affecting clinical and endoscopic efficacies of granulocyte, monocyte/macrophage in patients with ulcerative colitis.
Fifty consecutive patients with active ulcerative colitis initially received five granulocyte, monocyte/macrophage adsorptive apheresis sessions with the Adacolumn over five consecutive weeks. Patients who improved without achieving remission received five additional granulocyte, monocyte/macrophage adsorptive apheresis sessions.
One week after the last granulocyte, monocyte/macrophage adsorptive apheresis session, 26 (52%) and 17 patients (34%) achieved clinical and endoscopic remission, respectively. In the multivariate analysis, the dose of prednisolone administered at entry and the cumulative dose of prednisolone administered before entry were independent significant factors for both clinical and endoscopic remission, negatively impacted the efficacy of granulocyte, monocyte/macrophage adsorptive apheresis. Age, gender, duration of ulcerative colitis, number of prior relapses, duration of current exacerbation, extent and severity of ulcerative colitis, extra-intestinal manifestations, entry haematology values and C-reactive protein did not affect the outcome.
Based on the outcomes of this study, it appears that steroid-naïve patients and patients on low dose steroid and short duration of exposure respond to granulocyte, monocyte/macrophage adsorptive apheresis. Further studies in larger cohorts of patients should strengthen our findings.
Full-text · Article · Aug 2007 · Digestive and Liver Disease
[Show abstract][Hide abstract] ABSTRACT: An elevated serum remnant lipoprotein cholesterol (RLP-C)/triglyceride (TG) ratio has not been evaluated as an index of familial type III hyperlipidaemia defined by the presence of beta-VLDL and apolipoprotein (Apo) E2/2 phenotype in the Japanese hyperlipidaemic population.
Serum lipids and lipoproteins from 514 individuals (200 men and 314 women, mean age 58 years) with total cholesterol >6.22 mmol/L and TG between 2.26 mmol/L and 9.04 mmol/L, selected from 25,080 subjects visiting the clinics for health checkup were analysed for a possible relationship with familial type III hyperlipoproteinaemia.
Median RLP-C concentration and RLP-C/TG ratio were 0.30 and 0.11 mmol/L, respectively. When compared between subjects with (31 cases) and without (483 cases) a broad-beta band on electrophoresis, the RLP-C concentrations and RLP-C/TG ratio were 0.77 +/- 0.43 mmol/L versus 0.34 +/- 0.16 mmol/L (P<0.0001) and 0.15 +/- 0.023 versus 0.11 +/- 0.027 (P<0.0001), respectively. Three cases with broad-beta band positive (the presence of beta-VLDL) showed RLP-C/TG ratio greater than 0.23 and RLP-C greater than 0.78 mmol/L, suggestive of type III hyperlipoproteinaemia, despite a lack of characteristic Apo E2/2 homozygosity. Cases with Apo E/Apo CIII ratio greater than 1.0 were not detected in this study group.
Serum RLP-C concentration and RLP-C/TG ratio, together with Apo E/Apo CIII ratio appear to be useful for screening familial type III hyperlipidaemia in the Japanese hyperlipidaemic population.
No preview · Article · Jul 2007 · Annals of Clinical Biochemistry
[Show abstract][Hide abstract] ABSTRACT: We have previously demonstrated tumor-specific alpha1,2fucosylation, which is associated with resistance of tumor cells to anticancer treatment in human colorectal tumor tissues. By using the YB-2 monoclonal antibody, the resulting products have been identified as Y, Le(b), and H type 2 antigens in colorectal tumor tissues.
Immunohistochemical analyses of colorectal cancer tissues (74 specimens) were performed with a newly established mouse monoclonal antibody, YB-3 specifically recognizing H disaccharide (Fucalpha1,2Galbeta) structures, and anti-A, anti-B, YB-2, and anti-sialyl Lewis X (SLX) antibodies, together with the analyses of glycosyltransferases involved in the synthesis of ABH antigens in the same tissues.
The YB-3 antibody enabled us to detect colorectal tumors, particularly tumors in the distal large intestine and the rectum, with high sensitivity (74.3%) and specificity (100%). From immunohistochemical and enzymatic analyses of colorectal tissues, we found that once alpha1,2fucosylation had proceeded in tumor tissues, blood group A or B antigen was also synthesized in approximately half of the tissues of A or B blood type, but not in their normal tissues. A correlation of survival rate with immunostaining of tissues was found only by YB-3 antibody and not by anti-A, anti-B, or anti-SLX antibody.
As a predictor of postoperative prognosis of patients with colorectal cancer, immunodetection of alpha1,2fucosylated antigens with the YB-3 antibody seemed to be superior to blood groups A, B, or SLX antigen in colorectal tumor tissues.
[Show abstract][Hide abstract] ABSTRACT: Pilot studies have indicated a therapeutic role for an apheresis device (Adacolumn) that selectively adsorbs leukocytes in patients with inflammatory bowel diseases. It may also exert immunoregulatory effects contributing to its clinical efficacy. This study aimed to correlate the clinical response to leukocyte apheresis with the expression of key cytokines in mucosal tissue, in peripheral leukocytes, and in plasma.
Ten patients (seven with Crohn's disease and three with ulcerative colitis, median age: 31 years) with mild to moderately chronic activity were recruited to an open study. Patients were refractory to or had a relapse despite conventional treatment including azathioprine. Leukocyte apheresis was performed once a week for five consecutive weeks. Clinical efficacy was assessed on week 7 and after 12 months. Colonoscopy with multiple biopsies was performed at the start of the study and after 7 weeks for semiquantitative immunohistochemical analyses of cytokines. Cytokine levels in blood and the proportion of cytokine producing CD4+ and CD8+ lymphocytes were determined.
The apheresis procedures were well tolerated and no major adverse events were encountered. The median clinical activity score decreased from 12 to 7 on week 7 (P=0.031, n=9) and to 4 after 12 months (P=0.004, n=9). Five patients were in clinical remission at the 12th month. Tissue interferon (IFN)-gamma-positive T-cells decreased in clinical responders (P=0.027) after apheresis. In parallel, significantly lower levels of IFN-gamma-producing lymphocytes were detected in peripheral blood. IFN-gamma-positive cells in pretreatment biopsies completely disappeared or decreased in posttreatment biopsies sampled on week 7 in responders (P=0.027) and appeared to predict the maintenance of long-term remission or response after 12 months.
Leukocyte apheresis is a novel and safe nonpharmacological adjunct therapy that may prove useful in steroid refractory or dependent patients when conventional drugs have failed. Down-regulation of IFN-gamma in mucosal biopsies and in peripheral leukocytes may be a predictive marker for sustained, long-term response.
No preview · Article · Oct 2006 · International Journal of Colorectal Disease
[Show abstract][Hide abstract] ABSTRACT: Intraocular inflammation (uveoretinitis) is one major complication of Behcet's disease (BD) and responds poorly to drug therapy. This open prospective study was to assess the efficacy of selective granulocytapheresis in patients with refractory uveoretinitis of BD. Fourteen patients aged 20-56 years were treated. Granulocytapheresis was done with an Adacolumn filled with cellulose acetate leucocyte carries or beads that adsorb granulocytes and monocytes from the blood in the column. Each patient received 5 Adacolumn sessions at one session/week over 5 consecutive weeks. The study was designed to allow each patient to serve as his or her own control. The total numbers of ocular attacks (OA) were monitored for 6 months before and after 5 Adacolumn sessions. The number of OA (mean +/- SD) per patient for the 6 months before Adacolumn was 4.21 +/- 1.6 and for the 6 months post Adacolumn was 2.93 +/- 1.39 ( P = 0.0275). Nine patients (64%) improved and 5 did not change or worsened. Further, for a sub-group (n = 7) with duration of BD > or =5 years, the number of OA were 4.71 +/- 1.89 for the first 6 months and 2.29 +/- 1.38 for the second 6 months ( P = 0.0054). The corresponding values for a sub-group (n = 7) with duration of BD<5 years were 3.71 +/- 1.25 and 3.57 +/- 1.13, indicating that patients with long duration of BD are better responders. We conclude that granulocytapheresis might be effective and safe for patients with refractory ocular BD. Further studies are necessary to fully evaluate the clinical efficacy of granulocytapheresis for BD.
No preview · Article · Jul 2006 · Journal of Clinical Apheresis
[Show abstract][Hide abstract] ABSTRACT: To investigate the relationship between ulcerative colitis (UC) clinical activity index (CAI) and circulating levels of IL-1ra, IL-10, IL-6 and IL-18.
Blood levels of IL-1ra, IL-10, IL-6 and IL-18 were measured in 31 patients with active UC, the mean CAI was 11.1, ranging from 5-25; and 12 healthy individuals as controls. Patients were given granulocyte and monocyte adsorptive apheresis (GMA) with Adacolumn. Leucocytes which bear the FcgammaR and complement receptors were adsorbed to the column leucocytapheresis carriers. Each patient could receive up to 11 GMA sessions over 8 wk.
We found strong correlations between CAI and IL-10 (r = 0.827, P < 0.001), IL-6 (r = 0.785, P < 0.001) and IL-18 (r = 0.791, P < 0.001). IL-1ra was not correlated with CAI. Following GMA therapy, 24 of the 31 patients achieved remission and the levels of all 4 cytokines fell to the levels in healthy controls. Further, blood levels of IL-1ra and IL-10 increased at the column outflow and inflow at 60 min suggesting release from leucocytes that adhered to the carriers.
Full-text · Article · Jun 2006 · World Journal of Gastroenterology
[Show abstract][Hide abstract] ABSTRACT: Selective granulocyte and monocyte/macrophage adsorptive apheresis is to increase the turnover of infected leucocytes and has increased CD4+ T cells, which are necessary for actions of interferon-alpha on hepatitis C virus. Therefore, granulocyte and monocyte apheresis was to enhance the efficacy of interferon + ribavirin.
Fifteen patients, 12 had interferon resistant hepatitis C virus and 3 were interferon naive. Hepatitis C virus genotype was 1b in 11 and 2a in 4. The mean plasma HCV-RNA was 728.3 kU/mL and alanine aminotransferase was 107.5 U/L. Granulocyte and monocyte apheresis was with the Adacolumn, which contains carriers that adsorb granulocytes and monocytes/macrophages. After five consecutive granulocyte and monocyte apheresis sessions over 5 days, interferon daily 6 million units for 4 weeks, then three times/week for 20 weeks+ribavirin (600-800 mg per patient per day) were given and followed for another 24 weeks.
During granulocyte and monocyte apheresis, plasma HCV-RNA transiently fell by up to 55%. Similarly, incubation of blood with the Adacolumn carriers caused a significant fall in HCV-RNA. Four patients were unavailable for efficacy evaluation. In the other 11, alanine aminotransferase normalised and at 11 weeks, plasma HCV-RNA was negative; six of these (55%) maintained their remission during the follow up.
Granulocyte and monocyte apheresis appears to deplete extra-hepatic hepatitis C virus reservoirs and generate active complement opsonins, which contribute to hepatitis C virus killing. Additional mechanism(s) are also likely and need to be elucidated in future studies with larger cohort of patients.
No preview · Article · Aug 2005 · Digestive and Liver Disease
[Show abstract][Hide abstract] ABSTRACT: Serum alpha1-acid glycoprotein (AGP), an acute-phase protein secreted by the liver, carries alpha(1,3)-fucosylated structures on its 5 highly branched, N-linked sugar chains.
Serum AGP levels in patients with various types of malignancies (n=214 patients) were measured using an enzyme-linked immunosorbent assay with anti-AGP antibody. To investigate glycoforms that differed in their degree of branching and extent of fucosylation, serum AGP samples were analyzed by crossed affinoimmunoelectrophoresis (CAIE) with concanavalin A, and Aleuria aurantia lectin (AAL), and anti-AGP antibody.
A significant difference (P <0.001) in serum AGP levels was observed in preoperative patients compared with levels in the healthy control group, but the levels in individual patients did not reflect their clinical status. Conversely, it was found not only that the patterns of AGP glycoforms differed widely in the patient group compared with the healthy control group, but they also changed depending on each patient's clinical status. Furthermore, AGP glycoforms seemed to be appropriate markers of disease progression and prognosis according to follow-up studies of 45 patients during prolonged preoperative and postoperative periods.
Patients with advanced malignancies who had AGP glycoforms that contained highly fucosylated triantennary and tetraantennary sugar chains for long periods after surgery were likely to have a poor prognosis. However, patients who had AGP glycoforms without such changes were expected to have a good prognosis.
[Show abstract][Hide abstract] ABSTRACT: The treatment of inflammatory leg ulcers complicated by rheumatoid arthritis (RA), which are unresponsive to conventional care, can be frustrating. Furthermore, as granulocytes and monocytes (GM) are major sources of inflammatory cytokines, they have the potential to initiate and perpetuate inflammatory skin lesions. Accordingly, a recent study reported the remission of pyoderma gangrenosum following the reduction of activated peripheral blood GM by adsorptive apheresis (GMA).
In this clinical study, we applied GMA to three cases, each with one leg ulcer below the knee and RA. The ulcers had not responded to conventional therapy, including disinfection, dressing, and antimicrobials, and therefore were thought to represent inflammatory vasculitic lesions. GMA was performed using a column with a capacity of 335 mL, filled with cellulose acetate beads that selectively adsorb granulocytes and monocytes/macrophages (Adacolumn). Each patient received one GMA session/week for five consecutive weeks. The duration of one session was 60 min, with a flow rate of 30 mL/min.
The ulcers began to recede after two GMA sessions and, by the end of the fifth session, the ulcers in all three patients had healed. No recurrence has been observed up to the time of this report. The treatment was well tolerated and no severe side-effects were observed.
GMA, which depletes activated neutrophils and monocytes/macrophages, appears to be effective for inflammatory skin ulcers which do not respond to conventional medications.
No preview · Article · Nov 2004 · International Journal of Dermatology