Journal of Clinical Apheresis 20:171–184 (2005)
`
`Adacolumn for Selective Leukocytapheresis as a
`Non-Pharmacological Treatment for Patients With
`Disorders of the Immune System: An Adjunct or an
`Alternative to Drug Therapy?
`
`Abbi R. Saniabadi,1* Hiroyuki Hanai,2 Yasuo Suzuki,3 Toshihide Ohmori,4 Koji Sawada,5 Naoki
`Yoshimura,6 Yasushi Saito,6 Yuji Takeda,1 Kazuo Umemura,2 Kazunao Kondo,2 Yasuhiko Ikeda,2
`Ken Fukunaga,7 Mitsuyoshi Nakashima,8 Alberto Beretta,9 Ingvar Bjarnason,10 and Robert Lofberg11
`
`1Japan Immunoresearch Laboratories, Takasaki, Japan
`2Hamamatsu University School of Medicine, Hamamatsu, Japan
`3Sakura Hospital, Toho University, Sakura, Japan
`4Ageo Central General Hospital, Ageo, Japan
`5Fujimoto Hospital Medicine, Osaka, Japan
`6Chiba University School of Medicine, Chiba, Japan
`7Hyogo College of Medicine, Hyogo, Japan
`8Hamamatsu CPT, Hamamatsu, Japan
`9San Raffaele Hospital, Milan, Italy
`10GKT Medical School, London, United Kingdom
`11Karolinska Institute at the IBD Unit, Sophiahemmet, Stockholm, Sweden
`
`Inflammatory and/or autoimmune diseases like ulcerative colitis (UC) or Crohn’s disease (CD) are debilitating
`chronic disorders that poorly respond to pharmacological interventions. Further, drug therapy has adverse
`effects that add to disease complications. The current thinking is that disorders like inflammatory bowel disease
`(IBD) reflect an over exuberant immune activation driven by cytokines including TNF-a. Major sources of
`cytokines include myeloid leukocytes (granulocytes, monocytes/macrophages), which in IBD are elevated with
`activation behavior and are found in vast numbers within the inflamed intestinal mucosa. Accordingly, myeloid
`cells should be the targets of therapy. Adacolumn is filled with cellulose acetate beads that selectively adsorb
`and deplete myeloid cells and a small fraction of lymphocytes (FccR and complement receptors bearing cells).
`In one study, 20 steroid naive patients with moderate (n ¼ 14) or severe (n ¼ 6) UC according to Rach-
`milewitz despite 1.5–2.25g/day of 5-aminosalicylic acid received 6 to 10 Adacolumn sessions at 2 sessions/week.
`Efficacy was assessed 1 week after the last session. The majority of patients responded to 6 sessions, 17 (85%)
`achieved remission. In 2 of the 3 non-responders, CAI was 8 and 12 in 1; all 3 had deep colonic ulcers at study
`initiation. Decreases were seen in total leukocytes (P ¼ 0.003), % neutrophils (P ¼ 0.003), % monocytes (P
`¼ 0.004), an increase in lymphocytes (P ¼ 0.001), decreases in C-reactive protein (P ¼ 0.0002), and rises in
`blood levels of soluble TNF-a receptors I (P ¼ 0.0007), II (P ¼ 0.0045). In a separate study, a case with very
`severe steroid refractory UC who received up to 11 sessions responded well and avoided colectomy. Further,
`myeloid cell purging with Adacolumn has been associated with the release of IL-1 receptor antagonist, sup-
`pression of TNF-a, IL-1b, IL-6, IL-8, down-modulation of L-selectin and the chemokine receptor CXCR3. In
`conclusion, selective depletion of myeloid cells appears to induce anti-inflammatory effects and represents a
`non-pharmacological treatment for patients with active IBD. The treatment has a clear drug-sparing role.
`Changes in blood levels of inflammatory and anti-inflammatory factors are thought to contribute to the efficacy
`Ó 2005 Wiley-Liss, Inc.
`of this procedure. J. Clin. Apheresis 20:171–184
`
`Key words: inflammatory bowel disease; soluble TNF-a receptors; L selectin; Adacolumn; CXCR3; hepatocyte
`growth factor; IL-1 receptor antagonist
`
`INTRODUCTION
`
`This report describes new therapeutic applications
`of apheresis to specific inflammatory and/or autoim-
`mune diseases that are debilitating and present major
`challenges to drug therapy. The work is expected to
`highlight new dimensions of the clinical utility of
`apheresis.
`Ó 2005 Wiley-Liss, Inc.
`
`Presented at the World Apheresis Association 10th Congress
`hosted by the American Society for Apheresis at its 25th Annual
`Meeting, May 5–8, 2004, Miami Beach, Florida.
`*Correspondence to: Dr. A. Saniabadi, Japan Immunoresearch
`Laboratories, 351-1 Nishiyokote Machi, Takasaki 370-0021,
`Japan. E-mail: saniabadi@jimro.co.jp
`
`Published online in Wiley InterScience
`(www.interscience.wiley.com)
`DOI: 10.1002/jca.20046
`
`

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`
`Even in an era of modern medicine and inno-
`vation in biologic therapies, the layman’s perception
`of ‘‘treatment’’
`is taking an appropriate medicine
`that can stem out the disease or submitting to a
`surgical procedure;
`if the disease does not remit,
`then the physician’s judgment might be question-
`able. However, it is important to bear in mind that
`drug therapy, by its very nature, involves adding a
`foreign substance or substances to the body sys-
`tems. Since, for most diseases, treatment requires
`repeated dosing of medicines often over long peri-
`ods of time, the medicines, although initially effec-
`tive,
`can lead to the disease becoming drug
`dependent or drug resistant (refractoriness) together
`with side effects that can be unpleasant and serious
`(body’s
`reaction to a non-biological
`substance).
`Indeed, side effects of drug therapy often add to the
`disease
`complexity [1–7]. Hence, a therapeutic
`strategy based on a non-drug intervention, a cor-
`rection or support of the body’s natural processes,
`should have virtues not shared by a drug-based
`therapy. In line with this thinking, apheresis in-
`volves removing from the body or blood (as this is
`the most convenient site to access the body) a
`substance or substances that are toxic or are asso-
`ciated with a prevailing health disorder. Accord-
`ingly, the therapeutic efficacy of apheresis depends
`on both the selectivity of the device material to
`adsorb (or extract) the unwanted species and on the
`operator’s skills (the latter being of utmost impor-
`tance). Apheresis is not expected to induce depen-
`dency or refractoriness.
`Today’s selective removal of constituents of cir-
`culating blood to achieve a therapeutic effect by
`apheresis is reminiscent of the rather crude technique
`of bloodletting (phlebotomy) and its therapeutic
`application at the time of Hippocrates (460–377 BC)
`in Ancient Greece. The perception then was that
`disease reflected the presence of a disease-causing
`agent in the blood and bloodletting was to expel the
`disease. Bloodletting was routinely and extensively
`performed for diseases like inflammation, infection,
`and fever [8]. This practice was subsequently popu-
`larized by another Greek physician, Claudius Galen
`(129–203 AD) who worked in Rome and became a
`very well-respected authority on medicine for over
`1,500 years. It may just be the folly of the past, but
`it is difficult to imagine that this procedure would
`have been so widely practiced for such a long time if
`it had not been associated with efficacy.
`In the early 20th century, centrifugation was
`introduced for selective depletion of blood compo-
`nents to achieve therapeutic effects [9,10], and even in
`today’s antibody-based therapy targeting an appro-
`priate cytokine, chemokine, or an adhesion molecule,
`apheresis is considered a non-drug strategy for the
`
`management of immune disorders, to remove plasma
`components that might be involved in the pathogen-
`esis of a given disease [11,12]. Similarly, apheresis
`could involve selectively removing from blood specific
`immune cells that are inappropriately increased or
`activated and are promoting disease. Adacolumn,
`which is featured in this report, is an example of a
`system that can selectively deplete activated myeloid
`leukocytes
`(granulocytes, monocytes/macrophages)
`from peripheral blood [13].
`Strategies that selectively remove plasma com-
`ponents or blood cells as therapeutic interventions
`have generally been safe and not associated with
`any major side effect in a significant number of
`patients. Recently, apheresis has been considered an
`effective alternative to drug therapy or as an ad-
`junct to conventional drug therapy with a potential
`for reduction of standard medication dosages. An
`example is the Adacolumn leukocytapheresis ther-
`apy of patients with ulcerative colitis (UC) in which
`corticosteroids were discontinued or reduced to a
`minimum [13–19]. Therefore,
`recently one area
`where apheresis has been considered to have ther-
`apeutic application is inflammatory bowel disease
`(IBD), which includes UC and Crohn’s disease
`(CD). Patients with IBD or rheumatoid arthritis
`(RA) require large and multiple doses of more than
`one drug over long periods of time. The prolonged
`drug therapy required to control
`symptoms of
`chronic diseases can make the future treatment
`difficult [2,3,8]. Hence, apheresis might be a natural
`approach to promote disease remission, spare or
`reduce drugs [18,19], and avoid drug-induced com-
`plications.
`
`EFFICACY STUDIES IN SELECTED DISEASE
`TARGETS
`
`Objectives
`
`The principal objective of this report is to intro-
`duce the reader to specific clinical conditions that
`reflect immune dysfunctions associated with leuko-
`cytes and, therefore, in which selective leukocytaph-
`eresis with the Adacolumn has shown promising
`efficacy. However, it might be true to say that the
`disorders covered all have aetiologies that as yet are
`inadequately understood and,
`therefore,
`respond
`poorly to drug therapy. Hence, this report is broad
`based and is intended to highlight the therapeutic
`potential of apheresis as a non-drug medical inter-
`vention to achieve disease remission or as an effective
`adjunct to conventional drug therapy to enhance re-
`sponse and reduce drug dosage or spare patients from
`exposure to unsafe medications that are likely to be
`used in these conditions.
`
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`
`173
`
`Fig. 1. Scanning electron photomicrographs showing typical morphological changes in human leukocytes that selectively adhere to the
`Adacolumn cellulose acetate carriers during therapeutic leukocytapheresis. a: a low-power view (400·) showing a single layer of mostly
`neutrophils and monocytes covering the surface of the carrier; b: neutrophils are firmly adsorbed to the carrier and surface microvilli
`have disappeared; viewed at 10,000·; c: an activated monocyte/macrophage with disappearing surface microvilli viewed at 12,000·; d: as
`in c, but neutrophils are seen with long pseudopodia and fully shed surface microvilli (see reference [20] for leukocytes with surface
`microvilli). In the background, a few activated platelets are also seen (d).
`
`Selective Leukocytapheresis With the Adacolumn
`
`The general features of the Adacolumn have been
`described in an earlier publication [13]. It is an
`adsorptive
`carrier-based leukocytapheresis device
`designed for selective depletion of myeloid leuko-
`cytes (developed by Japan Immunoresearch Labo-
`ratories, Takasaki, Japan). The device is CE-marked
`(validated) by TUV (notified body) and is approved
`by the Japan Ministry of Health for the treatment of
`active UC. The column has a capacity of 335 mL
`and is filled with cellulose acetate beads of 2 mm in
`diameter as the column adsorptive leukocytapheresis
`carriers. The carriers are bathed in 130 mL of sterile
`saline until use when the column is primed with
`additional sterile saline and then with heparinized
`saline prior to use. The most common site of blood
`access is a venepuncture in the antecubital vein of
`one arm (inflow to the column) and return to the
`patient is via the antecubital vein in the contralateral
`arm. Scanning electron microscopy (SEM) reveals
`that
`the carriers selectively adsorb granulocytes,
`monocytes/macrophages (myeloid cells), and a small
`
`fraction of lymphocytes [13]. These are the leuko-
`cytes that express both the FccR and complement
`receptors. In Figure 1, specimen SEMs [20] are
`presented, while the major receptors that mediate
`this selective adhesion are illustrated in Figure 2.
`
`Leukocytapheresis as an Alternative to
`Corticosteroid in Patients With UC
`
`Twenty patients, aged 15–49 years, mean (±SD)
`clinical activity index (CAI) 8.8 ± 4.2; range 5–17 [21]
`were treated according to the design by Suzuki et al.
`[18]. UC was severe in 6 patients (CAI>11) and
`moderate in 14 patients (4 < CAI £ 11). Further, all
`patients were on 5-aminosalicylic acid (5-ASA, 1.5 to
`2.25 g/day) for at least 8 weeks prior to the initiation
`of leukocytapheresis therapy, but none was on any
`corticosteroid and remained off steroids during ther-
`apy. Each patient was to receive up to a maximum of
`10 sessions of 60 minutes at 2 sessions/week. Efficacy
`was assessed 1 week after the last session. The out-
`come is summarized in Figure 3. CAI fell to clinical
`
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`

`174
`
`Saniabadi et al.
`
`Fig. 2. Schematic presentations of FccR, complement activation fragments, and complement receptors involved in leukocyte adhesion to
`Adacolumn cellulose acetate leukocytapheresis carriers. Patients with autoimmune diseases have circulating immune complexes (IC) in
`their plasma; cellulose acetate adsorbs IgG and IC. Upon adsorption, the binding sites on IgG and IC become available for the FccRs on
`neutrophils and monocytes/macrophages. Further, adsorbed IgG and IC species generate complement activation fragments C3a, C4a,
`and C5a (solubles) and others. The opsonins, C3b/C3bi derived from complement also adsorb onto the carriers and serve as binding sites
`for the leukocyte complement receptors CR1, CR3 (Mac-1, CD11b/CD18), and CR4. FccRs are believed to influence CR3 as well [83].
`Accordingly, leukocyte adsorption to the cellulose acetate carriers is mediated by complement opsonins, FccRs, and the complement
`receptors on leukocytes. The expressions of complement receptors are common features of neutrophils and monocytes/macrophages;
`lymphocytes are not known to express complement receptors except on small subsets of B, T, and NK cells. Similarly, FccRs are not
`widely expressed on lymphocytes except on a small population of CD19+B and CD56+NK cells. These basic insights explain why the
`carriers adsorb granulocytes and monocytes/macrophages, but not significantly lymphocytes (see Discussion for further details).
`
`remission levels (CAI £ 4) in the majority of patients
`within 6 sessions, only 2 of 20 patients received
`10 sessions. At post-treatment, the mean CAI was 3;
`range 0–12 (P ¼ 0.0001) and 17 of 20 patients (85%)
`were in clinical remission. In 2 of the 3 non-re-
`sponders, CAI was 8 and 12 in 1, all 3 had deep co-
`lonic ulcers on endoscopy [21] at study initiation.
`There was also significant (P ¼ 0003) fall
`in a
`C-reactive protein (inflammation marker). At entry,
`total peripheral white blood cell count (WBC, · 103/
`lL) was 9.8 ± 1.0, range 5.9 to 22.5, showing a
`moderate elevation. The corresponding values mea-
`sured within one week after the last leukocytapheresis
`session were 7.0 ± 0.6, 3.5 to 15.3, respectively,
`showing a significant decrease (P ¼ 0.003). Simi-
`larly, there were falls in % neutrophils (P ¼ 0.003),
`% monocytes (P ¼ 0.004), and an increase in lym-
`phocyte counts (P ¼ 0.001) and % (P ¼ 0.001).
`During the leukocytapheresis
`therapy, only two
`
`incidences of transient mild headache were reported.
`In both cases, the headache receded within 3 hours
`without medication.
`
`Treatment of a Case With Severe Steroid
`Refractory UC
`
`Figure 4 shows colonoscopy photographs from the
`descending colon of a case with severe corticosteroid
`refractory UC who was treated by leukocytapheresis
`with Adacolumn without additional drug therapy. The
`patient was a 23-year-old female known to suffer from
`severe fulminating (disease continues in the presence of
`intensive medication) total colitis. The findings on
`admission included bloody diarrhoea 12–15 times/day,
`CAI 22, abdominal pain, fever (38.5°C), and fatigue.
`Figure 4A shows that after 10 days of very high-dose
`intravenous (iv) steroid, there was still strong inflam-
`mation, extensive pseudopolyps, and longitudinal ul-
`
`

`

`Selective Leukocytapheresis for Immune Disorders
`
`175
`
`leukocytapheresis has been associated with a dramatic
`down-modulation of CXCR3 (see below). A typical
`response in a patient with pyoderma G is presented in
`Figure 5. This followed several recent studies that re-
`ported improvements in patients with skin lesions
`following adsorptive myeloid cell purging with Ada-
`column [23,31–34]. The patient reported here was a
`19-year-old male who developed exacerbated CD
`symptoms complicated by pyoderma G. His major
`symptoms were severe anal pain, bloody diarrhoea
`more than 20 times/day, fever, and pyoderma lesions
`on his external ears, buttocks, and legs. Inflammation
`markers were: CRP 19.9 mg/dL, erythrocyte sedi-
`mentation rate (ESR) 55 mm/hr, and leukocyte count
`17.9 · 103/lL. We decided to implement selective
`leukocytapheresis for this patient based on a recent
`report of its efficacy in patients with CD [35]. Leuk-
`ocytapheresis with Adacolumn was performed at 90
`minutes per session, flow rate 40 mL/minute. Initially,
`the patient received one session/week for 5 consecutive
`weeks. This was followed by a significant improve-
`ment in the patient’s pyoderma lesions. These clinical
`results encouraged us to proceed with a second course
`of leukocytapheresis therapy. After the second 5
`weeks, skin lesions had re-epithelialized (Fig. 5) and
`colonoscopy showed no active ulcer, but ulcer scars
`covered with regenerated mucosa in the anal canal,
`rectum, and sigmoid colon with no anal pain. Stool
`frequency was 2–3/day, CRP 0.6 mg/dL, leukocyte
`count 4.7 · 103/lL, and normal ESR. The patient was
`discharged on maintenance therapy with oral ele-
`mental diet (2,400 kcal) and 3 g/day 5-ASA. These
`dramatic improvements in a patient with pyoderma
`associated with Crohn’s disease suggest that selective
`leukocytapheresis with the Adacolumn may be an
`effective therapy for inflammatory skin lesions.
`
`Selective Leukocytapheresis for the Treatment of
`Hepatitis C Virus Infection
`
`An estimated 170 million individuals in the world
`harbor HCV [36,37]. Currently, the most common
`treatment regimen comprises a combination of inter-
`feron-a (IFN-a) with Ribavirin (IFN/RBV). This
`regimen fails to eradicate HCV in the majority of
`patients harboring HCV genotype 1 [38,39]. Dysre-
`gulated HCV specific T-cell
`function (memory
`CD4+/CD8+ T-cells) is thought to be involved in
`both impaired cell-mediated immunity against HCV
`and resistance to anti-viral drug therapy [40–43].
`Accordingly, memory CD4+, CD8+ cells’ responses
`might determine the treatment efficiency. In view of
`this background, the rationale for selective leukocy-
`tapheresis in the treatment of HCV includes the fol-
`lowing: (1) Leukocytapheresis with Adacolumn both
`in patients with RA [13] and UC [18] and in this study
`
`Fig. 3. Changes of ulcerative colitis clinical activity index (CAI)
`and peripheral blood white cell (WBC) counts (presented as %)
`during Adacolumn adsorptive leukocytapheresis therapy of 20
`steroid naive patients with active UC. The data show a significant
`decrease in total WBCs, neutrophils, and monocytes, but the
`change in lymphocytes was in the opposite direction, a marked
`increase in both absolute count: 1,638 ± 38, range 634–3,376/lL
`at entry vs. 1,942 ± 39, range 1,001–3612/lL one week after the
`last session and % (19 vs. 30). Efficacy in terms of CAI was 85%
`(17 of 20 patients).
`
`cers. Figure 4B shows the same site after 5 Adacolumn
`leukocytapheresis sessions, but still inflammation is
`strong together with contact bleeding. Figure 4C shows
`the same site after 10 sessions, inflammatory pseudo-
`polyps have largely disappeared. This patient achieved
`remission and continues to be in remission with im-
`munosuppressants without steroid. However, the effect
`of time (5 weeks vs. 10 weeks) should not be ignored
`when comparing 5 sessions vs. 10 sessions.
`
`Effects on Inflammatory Skin Lesions
`
`Inflammatory skin lesions including pyoderma
`gangrenosum (G) and psoriasis are persistent skin
`disorders that are often associated with other immune
`disorders. For example, pyoderma G is seen in about
`2% of patients with IBD and can be an indication for
`surgery [2]. The prevalence is much greater in patients
`with Behcet’s disease [22,23]. They are referred to as
`the extra-intestinal or extra-systemic manifestations
`of the main inflammatory disorder.
`Inflammatory skin lesions contain large numbers of
`myeloid leukocytes together with T cells [24–27] and,
`therefore, selective leukocytapheresis should be an
`appropriate treatment in these conditions. Further,
`leukocyte accumulation in skin lesions is understood
`to be mediated by chemokine receptors like CXCR3
`and CXCR4 [26–30]. Hence, chemokine receptors
`might be appropriate targets for the treatment of skin
`lesions.
`In
`line with
`this
`view, Adacolumn
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`

`176
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`Saniabadi et al.
`
`Fig. 4. Colonoscopy photographs of descending colon from a 23-year-old female with severe steroid refractory ulcerative colitis treated
`with Adacolumn. A: After 10 days of very high-dose iv prednisolone, there is still strong inflammation, extensive pseudopolyps, together
`with deep longitudinal ulcers. B: The same site after 6 Adacolumn sessions, but still inflammation is strong together with pseudopolyps
`and contact bleeding. C: The same site after 11 sessions; inflammatory pseudopolyps have largely disappeared. This patient achieved
`remission and continues to be in remission with immunosuppressants without steroid.
`
`a: Pyoderma G lesions on the external ear of a 19-year-old male with Crohn’s disease. The patient was treated by selective
`Fig. 5.
`depletion of myeloid cells with the Adacolumn. b: After 6 sessions. c: Taken at week 12 after a further 4 sessions to ensure stable
`remission of both Crohn’s disease and pyoderma G.
`
`was followed by a significant increase in lymphocyte
`counts and a rise in CD4+/CD8+ T-cells [13]; (2)
`granulocytes and monocytes/macrophages are extra-
`hepatic replication sites and reservoirs for HCV dis-
`semination [44–46]; (3) the Adacolumn leukocytaph-
`eresis carriers (cellulose acetate) generate complement
`activation fragments, C3a, C5a, SC5b-9 complex, and
`the opsonin C3bi [13,47], which should contribute to
`HCV clearance via enhanced cell-mediated immunity
`and opsonization of viral particles. Accordingly,
`Adacolumn leukocytapheresis should be an effective
`non-pharmacological adjunct to IFN/RBV combi-
`nation therapy. In Figure 6, a typical response in a
`54-year-old female with HCV genotype 1b (the most
`resistant to IFN) is presented. This patient previously
`had received IFN/RBV therapy but had failed to
`achieve negative HCV. Figure 6 shows aspartate
`aminotransferase (AST), alanine aminotransferase
`(ALT; both are markers of liver damage), and HCV-
`RNA profiles. After 5 consecutive leukocytapheresis
`
`sessions over 5 days, IFN daily 6 million units for
`4 weeks, then 3 times/week for 20 weeks together with
`RBV (600 mg/day) were given and followed for
`another 24 weeks. Figure 6 shows that during
`leukocytapheresis (prior to IFN/RBV therapy), plas-
`ma HCV-RNA fell significantly, but could not be
`maintained. This initial fall is thought to reflect HCV
`elimination attributable mainly to complement.
`However, sustained negative plasma HCV was seen
`shortly after IFN/RBV therapy. HCV rebound was
`not seen during the 24-week follow-up.
`
`CHANGES IN PRO- AND ANTI- INFLAMMATORY
`FACTORS
`
`Down-Modulation of the Leukocyte Chemokine
`Receptor CXCR3
`
`Figure 7 shows down-modulation of the chemokine
`receptor CXCR3 on leukocytes following a 6-week
`adsorptive leukocytapheresis with Adacolumn. The
`
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`

`Selective Leukocytapheresis for Immune Disorders
`
`177
`
`Fig. 6. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), both markers of liver damage and HCV-RNA profiles in a
`54-year-old female with HCV genotype 1b during Adacolumn leukocytapheresis followed by interferon-a/ribavirin (IFN/RBV) com-
`bination therapy. After 5 consecutive Adacolumn sessions over 5 days, IFN daily 6 million units for 4 weeks, then 3 time/week for 20
`weeks together with RBV (600 mg/day) were given and followed for another 24 weeks. As shown, during leukocytapheresis (prior to
`IFN/RBV), plasma HCV-RNA fell significantly, but could not be maintained. This initial fall is thought to reflect HCV elimination
`attributable mainly to complement. However, sustained negative plasma HCV was seen shortly after IFN/RBV therapy. HCV rebound
`was not seen during the 24-week follow-up. On the time axis, AST, ALT, and HCV-RNA profiles during the first 5 days and 11 weeks of
`IFN/RBV therapy are shown.
`
`patient had refractory psoriasis that had not re-
`sponded to drug therapy over several years. The pa-
`tient was given 6 Adacolumn leukocytapheresis
`therapy at one session/week; the last session was on
`day 35. It can be seen that maximum down-modula-
`tion of CXCR3 occurred on day 70, well into the
`follow-up time. CXCR3 is a homing receptor selective
`of T-cells. Chemokines and their receptors regulate
`the migration of Th1, Th2 lymphocytes and myeloid
`cells to inflammatory lesions during the immune
`response [26–29]. In Figure 7, the level of CXCR3 seen
`at day 70 is within the normal range for all three major
`leukocyte populations.
`
`Leukocytapheresis Induced Changes in
`Leukocytes Adhesion Molecules
`
`To infiltrate tissues, granulocytes must first adhere
`to endothelial cells and then be able to transmigrate
`into tissues. Leukocyte rolling (tethering) that is fol-
`lowed by adhesion is mediated by LECAM-1
`(CD62L,
`leukocyte-endothelial cell adhesion mole-
`cule-1 or L-selectin) expressed on leukocytes [48–51].
`The leukocyte integrin, Mac-1 (CD11b/CD18),
`is
`expressed predominantly on monocytes/macrophages
`
`and granulocytes and to a higher extent when these
`cells are activated [48–50]. By using a published
`method [52], the expression of LECAM-1 and Mac-1
`on leukocytes in the blood at the Adacolumn inflow
`and outflow was investigated with flow cytometry.
`Figure 8 shows that in patients with RA (n ¼ 28),
`after selective leukocytapheresis, the expression of
`LECAM-1 on the unadsorbed leukocytes was down-
`modulated. In contrast, the expression of Mac-1 was
`up-regulated. On the basis of the aforementioned,
`down-modulation of LECAM-1 by leukocytapheresis
`can result
`in impaired leukocyte-endothelial cell
`interactions,
`leukocyte trafficking, and while LE-
`CAM-1 is down-modulated, up-regulation of Mac-1
`is not sufficient
`to promote leukocyte trafficking
`because the initial step (rolling) is down.
`
`Release of Soluble TNF-a Receptors During
`Adacolumn Leukocytapheresis
`Figure 9 shows elevated soluble TNF-a receptors
`I and II (s-TNF RI/RII) in the blood measured [53] at
`the Adacolumn inflow (at the start of leukocyta-
`pheresis) and outflow (at 60 minutes) and in the
`peripheral blood at 60 minutes. This set of data shows
`
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`178
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`Saniabadi et al.
`
`Fig. 7. Down-modulation of the chemokine receptor CXCR3 on peripheral blood leukocytes following a 6-week selective myeloid cell
`depletion with the Adacolumn. Treatment frequency was one session/week; the last session was on day 35. It can be seen that maximum
`down-modulation has occurred on day 70 well into the follow-up period. The level seen at day 70 is within the normal range for all three
`leukocyte sub-populations.
`
`Fig. 8. Flow cytometry recordings showing down-modulation of L Selectin expression and up-modulation of Mac-1 expression on
`leucocytes in the Adacolumn. Data from patients with rheumatoid arthritis (n ¼ 28). L selectin, Mac-1, and CXCR3 (Fig. 7) are
`adhesion and trafficking receptors for leukocytes; they are pro-inflammatory. Hence, down-modulation of L selectin and CXCR3 should
`impair leukocyte trafficking to tissues where they promote inflammation and injury. It should be stated that in the presence of L selectin
`being down, up-regulation of Mac-1 is not sufficient to enhance leukocyte trafficking.
`
`

`

`Selective Leukocytapheresis for Immune Disorders
`
`179
`
`Fig. 9. Elevated soluble TNF-a receptors I and II (s-TNF RI/RII) in the blood measured at the Adacolumn inflow (at the start of
`leukocytapheresis) and outflow (at 60 minutes) and in the peripheral blood at 60 minutes. This set of data show that s-TNF RI/RII in the
`peripheral blood increased during Adacolumn leukocytapheresis. The increase is believed to indicate release from myeloid leukocytes
`(granulocytes and monocytes) that adhere to the Adacolumn carriers. RI and RII have strong anti-inflammatory actions (see text). *P <
`0.05 vs. inflow.
`
`that s-TNF RI/RII at the column outflow and in the
`peripheral blood increases during Adacolumn leuko-
`cytapheresis. The increase is believed to indicate re-
`lease from leukocytes that adhere to Adacolumn
`carriers (myeloid cells shown in Fig. 1). The major
`cellular sources of s-RI/RII are thought to be neu-
`trophils and monocytes [47,54]; lymphocytes do not
`readily shed RI/RII [47]. RI and RII are known to
`have strong anti-inflammatory effects; they neutralize
`TNF without invoking TNF-like actions [55,56].
`
`Adsorption-Dependent Release of IL-1 Receptor
`Antagonist and Hepatocyte Growth Factor
`
`interleukin-1
`the release of
`Figure 10 shows
`receptor antagonist (IL-1ra) and hepatocyte growth
`factor (HGF)
`from granulocytes and monocytes
`upon adsorption to the Adacolumn cellulose acetate
`carriers. IL-1ra and HGF were measured by using
`enzyme-linked immunosorbent assays (ELISA) pre-
`cisely as described by Takeda et al. [57]. IL-1ra has an
`essential role in the control of inflammation in the
`mucosa [58,59] while HGF is known to promote
`mucosal epithelial cell regeneration, which is an
`essential step in ulcer healing [60].
`
`DISCUSSION
`
`UC and CD are debilitating chronic inflammatory
`bowel diseases that respond poorly to currently
`available
`pharmacological
`interventions
`with
`
`salicylates, immunosuppressants, corticosteroids, and
`even novel biologicals [2,3,61–63]. Indeed, repeated
`administrations of these agents, often at high doses
`over long periods of time can produce additional
`complications [1–7]. This indicates that currently the
`aetiology of IBD is inadequately understood and drug
`therapy is empirical rather than based on sound
`understanding of the disease mechanism. Nonethe-
`less, the present era of antibody-based therapy tar-
`geting specific cytokines, chemokines and adhesion
`molecules reflects some progress, though truly effec-
`tive in the minority of
`treated patients [61–63].
`Cytokines in particular represent the best validated
`therapeutic targets and it is logical to view IBD as a
`disorder reflecting an over-exuberant activation of the
`immune system driven by excessive generation of
`inflammatory cytokines like TNF-a, IL-1b, IL-6, and
`chemokines like IL-8, which lead to a persistent
`intestinal
`inflammatory response. Hence, cytokines
`and cell surface molecules expressed by activated
`specific immune cells are involved and represent po-
`tential
`targets
`for
`intervention. However, major
`sources of inflammatory cytokines include myeloid
`leukocytes, which in IBD are elevated with activation
`behavior, prolonged survival, and are found in vast
`numbers within the inflamed intestinal mucosa [2,64–
`66]. Indeed, neutrophil infiltration into the mucosal
`tissue can predict relapse of both UC and CD [64].
`This indicates that during clinical remission, myeloid
`leukocytes infiltrate the intestinal mucosa and have a
`major role in mucosal inflammation, injury, and IBD
`relapse [64–66]. Indeed, neutrophil activation and
`
`

`

`180
`
`Saniabadi et al.
`
`Fig. 10. Release of interleukin-1 receptor antagonist (IL-1ra) and hepatocyte growth factor (HGF) from granulocytes and monocytes
`upon adsorption to the Adacolumn leukocytapheresis carriers. As shown under identical conditions, no significant TNF-a or IL-1b
`could be measured. Similarly, the amounts of IL-1ra and HGF released were markedly increased in the presence of carriers. For
`measuring IL-1ra, TNF-a, and IL-1b, heparinized blood from healthy volunteers was incubated with or without carriers for 60 minutes
`as described by Takeda et al. [57]. For measuring HGF, plasma-free blood cells in RPMI 1640 medium were incubated for 60 minutes
`with the carries that had been pre-treated with 100% plasma [57]. The amounts of cytokines were measured by ELISA. Data are the
`mean ± S.E values for the numbers shown.
`
`prolonged survival is a feature of persistent inflam-
`mation and neutrophil-mediated mucosal damage has
`been shown to be associated with the development of
`IBD [65,66]. Accordingly,
`selective depletion of
`myeloid leukocytes by adsorptive apheresis with the
`Adacolumn has been associated with dramatic effi-
`cacy and a marked reduction of inflammatory cyto-
`kines produced by leukocytes (see below).
`Factors believed to contribute to neutrophil acti-
`vation and increased survival time in IBD include
`inflammatory cytokines [67] and, paradoxically, cor-
`ticosteroids [68], which are commonly given to most
`patients with active IBD. This, in part, can explain
`why UC and CD often show poor response to drug
`therapy and strengthens our assumption that myeloid
`leukocytes are involved in the exacerbation and per-
`petuation of IBD. Hence,