Oxidative stress and antibody levels to periodontal bacteria in adults: the Nagasaki Islands study.

OBJECTIVE: Periodontitis is a chronic inflammatory disease of the tissues supporting the teeth and is caused by subgingival plaque. Systemic increases in reactive oxygen species are involved in pathogenesis of periodontitis. This study addressed the relationship between levels of serum oxidative stress and antibodies against putative periodontopathic bacteria and their association with periodontal conditions, in a community-based study. SUBJECTS AND METHODS: Serum samples were measured for reactive oxygen metabolite (ROM) levels and anti-oxidant capacity. The serum levels of immunoglobulin G (IgG) antibodies to Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), and Eikenella corrodens (Ec) were determined by ELISA. RESULTS: The participants with greater clinical attachment loss had higher serum ROM levels and IgG antibody titers to Pg. Serum ROM levels were positively correlated with antibody titers to Pg, Pi, and Ec. When the participants with greater probing pocket depth and clinical attachment loss were used as the dependent variables, high ROM levels showed a statistically significant associations in multivariate logistic analyses; the adjusted odds ratios were 2.9 (95% confidence interval = 1.0-8.5) and 6.0 (95% confidence interval = 2.0-17.6), respectively. CONCLUSIONS: It was concluded that an increased oxidative stress may be detrimental to periodontitis in Japanese community-dwelling adults.

Relationship between Campylobacter rectus and periodontal status during pregnancy.

INTRODUCTION: In a previous study, we showed that the growth of Campylobacter rectus is stimulated by the presence of female sex hormones in the culture medium. In the present study, we examined the relationship between C. rectus levels in the saliva and the periodontal status of pregnant women. METHODS: Unstimulated whole saliva was collected from 22 pregnant and 15 non-pregnant women. Periodontal pocket depth (PD) and bleeding on probing (BOP) were recorded. A quantitative real-time polymerase chain reaction was performed to determine the concentrations of suspected periodontopathogenic bacteria in the saliva samples. In addition, the concentration of estradiol in the saliva samples was measured by enzyme immunoassay. RESULTS: The average age, number of teeth, and total number of bacteria in the saliva of subjects in both groups were similar. The percentage of sites with a PD = 4 mm and the salivary estradiol concentrations were significantly higher in pregnant women than in non-pregnant women. In addition, the percentage of BOP sites and the C. rectus levels in the saliva of the pregnant women tended to be higher than in non-pregnant women, although these differences were not statistically significant. There were positive correlations between C. rectus levels and estradiol concentrations, and between C. rectus levels and the percentage of sites with PD = 4 mm in the pregnant women. CONCLUSION: These results indicate that C. rectus levels are higher in the oral flora of pregnant women and that this may be associated with increased salivary estradiol concentrations. This may contribute to periodontal disease progression during pregnancy.

Serotype-specific polysaccharide of Streptococcus mutans contributes to infectivity in endocarditis.

Streptococcus mutans and other viridans streptococci have been implicated as major etiological agents of infective endocarditis. The serotype-specific rhamnose-glucose polysaccharide (RGP) of S. mutans has several biological functions that appear to be essential for the induction of infective endocarditis. The aim of this study was to examine the contribution of RGP to the infectivity of S. mutans in infective endocarditis using a rat model. The RGP-defective mutant of S. mutans showed reduced ability to induce infective endocarditis compared to the parental strain. The ability of S. mutans to induce infective endocarditis was not consistent with the binding capacity of the organism to extracellular matrix proteins. The results suggest that S. mutans containing whole RGP is more virulent than the RGP-defective mutant, and the RGP has an important role for the induction of infective endocarditis by S. mutans.

Porphyromonas gingivalis-specific IgG subclass antibody levels as immunological risk indicators of periodontal bone loss.

BACKGROUND: It has been well demonstrated a positive association between the magnitude of host antibody response and periodontal disease status. Previous studies also reported that Porphyromonas gingivalis-specific IgG subclass antibodies were elevated in sera from adult periodontitis patients. However, the rôle and the association of these IgG subclass antibodies to the development of periodontal diseases are poorly understood. AIM: The aim of present investigation was to examine the relation of serum IgG subclass antibody levels and alveolar bone loss in treated and untreated periodontitis patients. METHODS: Serum samples were taken from 20 treated and maintained periodontitis patients (SPT patients), 30 untreated patients and 19 periodontally healthy subjects. We determined the IgG subclass antibody titers to P. gingivalis whole cells using an enzyme-linked immunosorbent assay (ELISA). Subgingival plaque samples were taken from the mesio-buccal surface of 6 randomly selected teeth of SPT patients and evaluated for the presence of P. gingivalis by immunofluorescence microscopy. Clinical measurements were also taken including full mouth intraoral radiographs to measure interproximal alveolar bone loss at baseline (BLS1) and at a 5-year recall visit in the SPT patients (BLS2). RESULTS: Our results indicated that both patient groups had detectable levels of IgG1, IgG2, and IgG4. Significantly higher IgG1 was observed in both patient groups compared to the healthy subjects. The untreated patients also exhibited significantly elevated IgG2 response (p<0.05). The mean IgG4 level of the SPT patients was significantly higher compared to the other subject group (p<0.05). A statistically significant positive correlation between IgG2 levels and changes in bone levels (DeltaBLS: BLS2-BLS1) was seen in the SPT patients (p<0.001). SPT patients with high IgG2 and low IgG4 showed greater bone loss than those with low IgG2 and high IgG4 (p<0.05), although the mean prevalence of P. gingivalis in the 2 groups did not differ significantly. CONCLUSION: Our data suggest that the prolonged IgG2 response after periodontal treatment may be indicative of recurrent or persistent periodontal destruction.

Periodontal status and serum antibody titers for Porphyromonas gingivalis fimbriae in a rural population in Japan.

BACKGROUND, AIMS: The present study was undertaken to assess the periodontal status of a rural Japanese population and to study the correlation between the periodontal status and the serum antibody titers for Porphyromonas gingivalis (Pg) fimbriae. METHOD: A total of 236 individuals were examined for their periodontal conditions by the use of the community periodontal index for treatment needs (CPITN), and serum antibody titers for Pg fimbriae in their peripheral blood samples were evaluated using the enzyme-linked immunosorbent assay. RESULTS: There was a substantially larger proportion of edentulous subjects in the age group older than 60 years. The remaining teeth were 24.1, 23.2, 11.1 and 10.1 per person in the 40-49, 50-59, 60-69 and > or = 70 age groups, respectively. The % of sextants with a CPITN code of missing sextant (MS) increased towards elderly and reached >60% in the age group of > or = 70 years, as the % of the CPITN 2, 1 or 0 sextant decreased. The % of CPITN 4 and 3 sextants did not differ between different age groups and were about 6-8% and 15-20%, respectively. The % of CPITN 1 or 0 sextants was higher in female subjects than in male subjects in the 60-69 and > or = 70 age groups, while the % of CPITN 4 or 3 sextants was higher in male subjects than in female subjects in all age groups. There was no significant difference between various age groups in the mean serum antibody titers for Pg fimbriae. The mean anti-Pg fimbriae antibody titers was significantly higher for the subjects with a maximum CPITN code 4 (max.-CPITN 4 subject) than for the subjects with lower maximum CPITN codes. The antibody titers varied extensively among the max.-CPITN 4 or 3 subjects, but not among the max.-CPITN 2/1/0 or MS subjects. CONCLUSIONS: The present study demonstrated that tooth loss is a remarkable event in elderly subjects and that oral prophylaxis and mechanical debridement should be mandatory in the population examined. It was also demonstrated that the serum antibody titers against Pg fimbriae could be useful for screening individuals with moderate to severe periodontitis.

Endocardiac infectivity and binding to extracellular matrix proteins of oral Abiotrophia species.

Microorganisms of the genus Abiotrophia, formerly known as nutritionally variant streptococci, are members of the oral flora and often isolated from patients with endocarditis, but pathogenicity of oral Abiotrophia species has not been examined yet. In this study, 17 strains isolated from healthy human oral cavities and 7 reference strains (all derived from patients with endocarditis) of Abiotrophia spp. were tested for their abilities to cause infections in damaged heart tissues in catheterized rats and to adhere to extracellular matrix proteins in vitro. The reference strains of A. defectiva and A. adiacens showed high infectivities in the rats. Four oral isolates of these two species showed similarly high infectivities and three had moderate infectivities. Most of 10 oral strains of A. para-adiacens and A. elegans were found to be generally less infective. The highly infective A. adiacens strains showed markedly high fibronectin-binding capacity, suggesting a possible relationship between the fibronectin-binding capacity and damaged heart tissue infectivity of the Abiotrophia species. A. defectiva strains which were also highly infective had moderate levels of binding to fibronectin and other extracellular matrix proteins. Most of A. para-adiacens and A. elegans strains showed low or negligible binding capacities to any extracellular matrix proteins tested.

The molecular weight ratio of monomethoxypolyethylene glycol (mPEG) to protein determines the immunotolerogenicity of mPEG proteins.

Immunotolerogenic activity of monomethoxypolyethylene glycol- (mPEG) conjugated proteins is a beneficial property in protein pharmaceutics. However, procedures for the preparation of tolerogenic mPEG proteins have not yet been defined. We prepared mPEG proteins with different mPEG contents using three proteins, hen egg lysozyme, ovalbumin and bovine gamma globulin, and their tolerogenicities to antigen-specific T and B cell responses were examined. We found the most appropriate ratio of tolerance induction to be 1.5-2.0, which is the molecular weight ratio of conjugated total mPEGs to protein. This value may assist in the preparation of tolerogenic mPEG proteins.

Extended blood half-life of monomethoxypolyethylene glycol-conjugated hen lysozyme is a key parameter controlling immunological tolerogenicity.

The blood half-life of a protein is prolonged by conjugating a protein with a linear amphiphilic polymer, monomethoxypolyethylene glycol (mPEG). The conjugation gives a protein immunotolerogenicity; hence, it is likely that the long half-life is crucial for the tolerogenicity. We prepared a tolerogenic mPEG conjugate of hen egg lysozyme (mPEG1.5-HEL), which is conjugated 1.5-fold the molecular weight of mPEG against that of HEL, and evaluated the relationship between in vivo stability and the tolerogenicity. mPEG1.5-HEL retained immunogenicity to prime HEL-specific T cell and antibody responses and had a long blood half-life, more than 27 times that of native HEL. The tolerant state was maintained as long as mPEG1.5-HEL was detected in sera. With a decrease in the blood mPEG1.5-HEL level, the tolerant state returned gradually to the responsive state; however, reinjection of mPEG1.5-HEL again restored the tolerance. Thus, the extended blood half-life of HEL by mPEG conjugation is probably vital for establishing and maintaining the tolerant states.

Nasopharyngeal-associated lymphoreticular tissue (NALT) immunity: fimbriae-specific Th1 and Th2 cell-regulated IgA responses for the inhibition of bacterial attachment to epithelial cells and subsequent inflammatory cytokine production.

To investigate the antibacterial activity of mucosal Th1 and Th2 immune responses induced nasally and orally, mice were immunized with mucosal vaccine containing fimbrial protein of Porphyromonas gingivalis, a causative agent for a destructive chronic inflammation in the periodontium, and cholera toxin (CT) as mucosal adjuvant. Nasal vaccine containing low doses of fimbriae (10 micrograms) and CT (1 microgram) induced Ag-specific Th1/Th2-type response in CD4+ T cells in mucosal effector tissues, including nasal passage and submandibular glands, which accounted for the generation of Ag-specific IgA-producing cells. In contrast, oral immunization required higher amounts of fimbriae and CT for the induction of Ag-specific IgA responses. Fimbriae-specific IgA mAbs generated from submandibular glands of nasally immunized mice inhibited P. gingivalis attachment to and reduced subsequent inflammatory cytokine production from epithelial cells. These findings suggest that nasal vaccination is an effective immunization regimen for the induction of Ag-specific Th1 and Th2 cell-driven IgA immune responses that possess the ability to inhibit bacterial attachment to epithelial cells and subsequent inflammatory cytokine production.

Tolerogenic activity of polyethylene glycol-conjugated lysozyme distinct from that of the native counterpart.

Conjugation of proteins with polyethylene glycol (PEG) has been reported to make the proteins tolerogenic. Native proteins are also potentially tolerogenic when given without adjuvants. We compared immunotolerogenic activities between PEG-conjugated and native hen egg-white lysozyme (HEL). BALB/c mice were given consecutive weekly intraperitoneal administrations of PEG-conjugated HEL, unmodified HEL or phosphate-buffered saline (PBS), for 3 weeks, then challenged with HEL in Freund's complete adjuvant. The pretreatment with PEG-HEL tolerized both T-cell and humoral responses, while native HEL tolerized only the T-cell response. Immunoglobulin G1 (IgG1) antibody was already elevated in HEL-pretreated mice prior to challenge immunization, followed by suppressed IgG2a and IgG2b, but spared IgG1 production after the antigen challenge, whereas PEG-HEL-pretreated mice produced no antibody in all IgG subclasses prior and subsequent to the antigen-challenge. Production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by lymphoid cells in response to HEL in vitro was markedly suppressed in both the antigen-pretreated groups, while suppression of IL-4 production was evident only in PEG-HEL-, not in HEL-pretreated animals. Involvement of suppressor cells in these tolerance states was found to be unlikely, and the immunological property of PEG-HEL differed from deaggregated HEL that was similar to the original HEL. These results suggest a unique immunotolerogenic activity of PEG-conjugated proteins to suppress both T-helper type-1 (Th1)- and Th2-type responses, the result being extensive inhibition of all IgG subclass responses, while tolerance induction by unconjugated soluble proteins tends to be targeted on Th1-, but spares Th2-type responses.

A novel approach for detecting an immunodominant antigen of Porphyromonas gingivalis in diagnosis of adult periodontitis.

In the course of long-term infection with Porphyromonas gingivalis in adult periodontitis, a specific antibody response to this organism is generated. We describe a potential novel approach for identifying an immunodominant antigen in human periodontitis patients. First, various monoclonal antibodies (MAbs) were established from mice immunized with crude antigen preparations of P. gingivalis FDC 381. The antigen specificities of these MAbs were compared with those of serum antibodies of 10 periodontitis patients in a competitive enzyme-linked immunosorbent assay. The binding of one MAb (termed PF18) was readily inhibited by sera from all patients but not by sera from healthy volunteers. The antigen recognized by PF18 existed on the cell surface, presumably in the capsule layer, shown by immunoelectron microscopic analysis. Purification of the antigenic substance, termed PF18-Ag, was performed by immunoaffinity chromatography with the MAb. Characterization of PF18-Ag suggested that the epitope was composed of carbohydrates but not peptides and that the substance was different from lipopolysaccharide. Measurement of levels of serum antibody to PF18-Ag better discriminated periodontitis patients from healthy individuals than measurement of antibodies to crude antigen preparations of P. gingivalis. Immunoglobulin G2 was the predominant isotype among the antibodies to PF18-Ag in the patients' sera. These results suggest that PF18-Ag, which is possibly a novel substance, is an important antigenic substance and is potentially useful for the clinical diagnosis of adult periodontitis. The approach that was used would also be relevant to detecting immunodominant antigens of other infectious microorganisms.

Prevention of collagen-induced arthritis (CIA) by treatment with polyethylene glycol-conjugated type II collagen; distinct tolerogenic property of the conjugated collagen from the native one.

Administration of a soluble protein into animals prior to challenge immunization induces immunological tolerance which is specific for the protein. In addition, chemical modification of proteins with polyethylene glycol (PEG) has been reported to convert the immunogenic proteins to become tolerogenic. However, differences in tolerogenic properties between PEG-modified proteins and the native counterparts have never been analysed. The ability of PEG-conjugated type II collagen (PEG-CII) to attenuate CIA, an animal model for rheumatoid arthritis, was compared with the native unconjugated CII. Groups of DBA/1 J mice were treated weekly with i.p. injections with PEG-CII, native CII, or vehicle alone for 3 weeks, before they were challenged with CII in adjuvants. The induction of tolerance was confirmed in both PEG-CII- and CII-pretreated mice when suppression of lymph node T cell proliferation in response to CII was noted. The degrees of suppression of T cell proliferation were comparable between the two pretreated groups. However, induction of arthritis and production of IgG anti-CII antibody were more markedly suppressed in PEG-CII-pretreated mice than in native CII-pretreated mice, although the severity of arthritis and antibody levels in the latter group were also lower than in control mice. IgG2a and IgG2b antibody levels were equally suppressed in the two pretreated groups, whereas the IgG1 level was significantly lower in the PEG-CII-pretreated group than in the native CII-pretreated group. The results provide the first evidence that attachment of PEG to CII renders the protein more tolerogenic.

Role of the charged tail in localization of a surface protein antigen of Streptococcus mutans.

To make clear the role of the C terminus of a surface protein antigen (PAc) of Streptococcus mutans, stepwise truncations beginning at the C terminus of PAc were performed by utilizing site-directed mutagenesis. A remarkable increase in the amount of cell-free PAc was observed upon deletion of four or more amino acid residues at the C terminus. On the other hand, the amount of cell surface PAc gradually decreased when increasing numbers (four or more) of amino acid residues were deleted at the C terminus, and deletion of six amino acids involving both the total charged tail and Leu, an amino acid residue immediately upstream of the charged tail, resulted in a drastic reduction in the amount of cell surface PAc. These results indicate that the cytoplasmic charged tail and Leu residue are required for cell surface localization of PAc in S. mutans.

Hen egg-white lysozyme inhibits biological activities of lipopolysaccharides from periodontopathic bacteria.

Lysozyme has a bactericidal activity for some strains of Gram-positive bacteria, by enzymatic cleavage of peptidoglycans that constitute the cell wall. Hen egg-white lysozyme (HEL) was tested in vitro for effects on biological activities of lipopolysaccharides from periodontopathic Gram-negative bacteria. Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromonas gingivalis. HEL inhibited a wide range of activities of these lipopolysaccharides, including activation of Limulus amoebocyte lysate, stimulation of human leukocytes to secrete tumour necrosis factor-alpha, polyclonal activation of mouse B cells, and promotion of osteoclastic differentiation in mouse bone marrow cultures. The anti-endotoxic activity of HEL may be worthy of being intended for periodontal therapy.

Quaternary structure-dependent idiotope and antigen binding of a monoclonal antibody specific for conformational epitope on type II collagen.

We previously generated a monoclonal antibody (mAb) against a putative pathogenic epitope on native type II collagen (CII) for the induction of collagen-induced arthritis in mice (mAb1), and an anti-idiotypic mAb which appears to possess the internal image of the CII epitope (mAb2). In the present study, the structural basis of the antigen/mAb1 and mAb1/mAb2 interactions was examined. When partially SH-reduced mAb1 was analysed on Western blots, only fragments containing both heavy (H) and light (L) chains were recognized by mAb2. When mAb2 was partially SH-reduced, only fragments containing both H and L chains were recognized by mAb1. H and L chains were separated from mAb1 in a reduced, denatured condition, and each chain and a mixture of the two were refolded. mAb2 reacted specifically to the renatured whole IgG molecule of mAb1, but not to the refolded L or to H chains. Recombinant single chain Fv (scFv) generated from mAb1 and mAb2 had properties of the original mAbs, whereas genetically constructed chimeric scFvs, consisting of VH from mAb1 and an irrelevant VL. or VL of mAb1 and an irrelevant VH. did not react either to CII or to mAb2. Thus, interactions among CII, mAb1 and mAb2 appear to depend on quaternary structures containing different protein subunits. These observations support the internal image property of the mAb2. In addition, this dependency on quaternary structure for recognition of proteins may also be relevant to other protein-protein interactions.

Depression of T-cell epitope generation by stabilizing hen lysozyme.

Conformational stability of proteins is an important factor that determines their resistance/susceptibility to proteolytic digestion. Intracellular proteolysis is the key step in antigen presentation events for protein antigens; hence, it is likely that increasing protein stability reduces the antigenicity of proteins. We prepared three hen egg white lysozyme derivatives possessing different stabilities by chemical modification to clarify the relationship between conformational stability and the antigenicity of the protein. One of the derivatives was conformationally unstabilized by removing one intramolecular disulfide bond, whereas the two others were stabilized by the addition of an intramolecular cross-link. The antigenicity of these derivatives was evaluated using hen egg white lysozyme-specific T-cell hybridoma cells and a B-lymphoma cell line, A20, as antigen-presenting cells. With an increase in conformational stability, the T-cell response decreased. However, the reduction was not derived from the inefficiency of internalization to A20 cells nor the alteration of antigenicity by chemical modifications. Moreover, from analyses of their susceptibility to proteolysis and the kinetics of presentation of the T-cell epitope, it was confirmed that increasing protein stability led to the depression of T-cell epitope generation by increasing resistance to proteolysis. These results have an important implication in devising a new strategy for manipulating T-cell response by the stability of protein antigen.

Lipopolysaccharides from Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans promote osteoclastic differentiation in vitro.

Bacterial lipopolysaccharides possess bone-resorbing activity. Here, lipopolysaccharides from three putative periodontopathic bacteria were examined for effects on osteoclast-like cell formation of bone marrow cells from lipopolysaccharide-responsive C3H-HeN and non-responsive C3H/HeJ mice. The bone marrow cells were cultured with or without various doses of lipopolysaccharide in the presence of 1,25-dihydroxyvitamin D3 and dexamethasone. These lipopolysaccharide preparations significantly increased the number of osteoclast-like cells formed in the culture of C3H/HeN marrow cells; the same as lipopolysaccharides from Escherichia coli and a synthetic lipid A with E. coli-type structure (LA-15-PP), at doses from 0.1 to 1 microgram/ml. This stimulating effect of each lipopolysaccharides was uniformly abrogated by the addition of polymyxin B at 5 micrograms/ml. All the lipopolysaccharide and the synthetic lipid A had no effect on osteoclast formation of the C3H/HeJ marrow cells, whereas lipopolysaccharide from Porphyromonas gingivalis and Prevotella intermedia showed significant mitogenic activity on C3H/HeJ spleen cells. It seems likely that the activity of lipopolysaccharides to augment osteoclast-like cell formation in the bone marrow cell cultures is derived from a common structure of the lipid A portion.

Reduced immunogenicity of monomethoxypolyethylene glycol-modified lysozyme for activation of T cells.

Chemical modification of proteins with monomethoxypolyethylene glycol (mPEG) will reduce the immunogenicity of proteins. In the present study, we evaluated the effect of mPEG modification on the capacity of hen egg-white lysozyme (HEL) to stimulate T cells. Lymph node cells (LNCs) from mice immunized with HEL or with mPEG-HEL conjugate were cultured with these antigens, then we measured the proliferation and IL-2 production. mPEG-modification lowered the T cell activating capacity of HEL, both in vitro and in vivo. Neither toxicity, nor antigen non-specific immunosuppressive capacity was observed with mPEG-HEL and unconjugated mPEG. Suppressor cells were unlikely to be generated in the mPEG-HEL-primed LNCs. We next examined the behavior of mPEG-HEL during antigen processing. The capacity of HEL and mPEG-HEL to be incorporated by live cells was much the same. However, the susceptibility to various proteases, including endosomal/lysosomal enzymes, was significantly decreased by mPEG modification. The increased resistance of mPEG-HEL to proteolytic degradation implied that the conjugate was poorly presented to T cells. This may be an important factor related to the low immunogenicity of mPEG modified proteins.

An immunotoxin system intended for bone marrow purging composed of glucose oxidase and lactoperoxidase coupled to monoclonal antibody 097.

The effectiveness of an antibody-enzyme immunotoxin (eIT) was investigated on human T cells. This enzyme immunotoxin contained glucose oxidase (GO) and lactoperoxidase (LPO) chemically coupled to the pan-leukocyte-specific mouse monoclonal antibody (MoAb) 097 (097-GO and 097-LPO). Human peripheral blood mononuclear cells or tumor cells were suspended in a mixture of 097-GO and 097-LPO for 30 min, and then for 2 h with glucose and NaI. The effectiveness of this eIT system was indicated by the almost complete reduction of T cell viability, as estimated by a phytohemagglutinin induced proliferation assay (99.4% +/- 0.31 depletion, mean +/- SEM of 15 experiments). The specificity of the cytotoxicity reaction was indicated by the lack of cytotoxicity of control irrelevant MoAb conjugates to T cells (1.9% +/- 4.17 of T cell depletion, eight experiments). The growth of human bone marrow myeloid progenitors (CFU-GM) was not affected by the conjugates even by increasing 100-fold the optimal cytotoxic dose. T cells were susceptible to the conjugates in the presence of up to 90% of erythrocytes. This eIT system may thus represent a new alternative immunospecific procedure for allograft and/or autograft purging, and appears to effectively replace complement-mediated methods of T cell depletion.