Lymphocyte Adhesion Molecules Research Articles

Salmonellosis as a Complication of Natalizumab Therapy in a Patient with Ulcerative Colitis and Multiple Sclerosis

Purpose:Salmonella enterica is a gram negative enteric pathogen which is acquired via oral ingestion of contaminated food or water. Diarrheal disease caused by non-typhoidal salmonella serovars (NTS) such as Salmonella enteritidis results in a colitis that mimics ulcerative colitis (UC). The use of biologic therapy in patients with inflammatory bowel disease leads to late and relapsing Salmonella infection. However, to our knowledge there are no reports of patients who develop Salmonella infection while on selective adhesion molecule (SAM) inhibitor class therapy. A 49-year-old woman with well-controlled left sided UC over the last two decades and an 11-year diagnosis of MS presented with new onset abdominal cramping, non-bloody diarrhea, rash, and pain in her wrists, hips, and elbows. She had been on colazal 6.75 g daily and Natalizumab (NTZ) 300 mg monthly for the last 3 years. Her vital signs were stable. She was in no acute distress. Her abdominal exam was unimpressive but her skin was remarkable for several rose colored papules on her chest and flank. Laboratory data was significant for a creatinine of 1.1 mg/dL, a WBC of 11,200/uL, and ESR of 30 mm/h. Stool studies were positive for Salmonella enteritidis. NTZ was discontinued for four months following this diagnosis. The patient was treated with rifaximin 200 mg po TID for 10 days with resolution of her symptoms in 5 days. She experienced a culture positive relapse 3 weeks later and was again treated with rifaximin but for an extended duration. NTZ is a recombinant humanized IgG4 monoclonal antibody that blocks α4 integrin, a lymphocyte adhesion molecule. It prevents marginalization of inflammatory cells from the bloodstream into inflamed tissues. IL-8 produced by enterocytes infected with Salmonella is responsible for the chemotaxis of macrophages, the most efficient effector cell in clearing Salmonella infection. Disruption of macrophage transmigration by NTZ will thereby reduce the host's ability to clear Salmonella. In immunocompetent adults, diarrheal disease caused by NTS is typically self-limiting and antibiotic treatment may actually lead to a prolonged shedding phase, relapse of disease, and increased risk for adverse drug reactions. Antibiotic treatment is recommended in those patients with severe illness or who are immunosuppressed with evidence for bacteremia, focal or suppurative infections. Here we report the first case of a relapsing NTS infection in a patient with a history of UC and MS on NTZ and recommend antibiotic treatment in similar cases given the impairment of the macrophage effector arm in this infection.

Decreasing High Failure Rate of Vaccinations in Patients With Chronic Kidney Disease; not Just a Matter of Quantity

Dear Editor, In their recent interesting published manuscript, Ahmadi et al. showed that in predialysis patients with chronic renal failure (CRF) doubling the dose of Hepatitis B virus (HBV) vaccine from 20mcg to 40mcg was not benefit regarding the seroconvertion rate [1]. Although somewhat provocative, the above highlights the complexity of the immune system and the difficulties in interfering effectively and safely with its function. In CRF patients the impaired immune function is multifactorial. The impaired interaction between antigen presenting cells (APCs) and T-cells is indicated by the decreased T-cell proliferation and ζ-chain phosphorylation after a major histocompatibility complex (MHC) dependent T-cell receptor (TCR) stimulation and certainly contributes to the impaired immune function. Decreased MHC and TCR expression, altered expression of the adhesion molecules lymphocyte function antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1), as well as reduced expression of the co-receptors CD80/CD86 in APCs and CD28 in T-cells may be responsible for it. Besides the impaired APC-T-cell interaction; other mechanisms have been also proposed for the acquired immunity disturbances in CRF patients. Increased soluble CD40 molecules, which inhibit the interaction between T-cells and B-cells, decreased TCR complex ζ-chain expression, altered cytokine status, which affects T-cell differentiation and finally increased apoptosis of T-cells and B-cells have all been incriminated for the impaired adaptive immune response [2]. Recently, our group showed that the immunomodulatory enzyme indoleamine 2, 3-dioxygenase is increased in CRF patients and negatively affects the immune response to HBV vaccine [3]. The above complexity indicates that the hypo responsiveness to HBV vaccine, which is observed in CRF patients, cannot be overcome simply by increasing the dose of the antigen. The solution could be in the development of new vaccines with adjuvants other than the aluminum hydroxide, which is generally used. For example, an approved vaccine against HBV for CRF patients contains mono phosphoryl lipid A (MPL). MPL is a low-toxicity derivative of lipopolysaccharide that interacts with Toll-like receptors and the mentioned vaccine showed improved immunogenicity when administered in CRF patients [4]. Recent advances in immunology revealed that early innate immune signals shape subsequent adaptive responses and this has led to the design and development of more specific and focused adjuvants [5]. Certainly, safe and more effective vaccines will be available soon for CRF patients, which are characterized by impaired adaptive immune response.

Enteral n-3 fatty acids and micronutrients enhance percentage of positive neutrophil and lymphocyte adhesion molecules: a potential mediator of pressure ulcer healing in critically ill patients

n-3 Fatty acids are recognised as influencing both wound healing and immunity. We assessed the impact of a fish oil- and micronutrient-enriched formula (study formula) on the healing of pressure ulcers and on immune function in critically ill patients in an intensive care unit. A total of forty patients with pressure ulcers and receiving nutritional support were enrolled (intervention group, n 20, received study formula; and a control group, n 20, received an isoenergetic formula). Total and differential leucocyte count and percentage of adhesion molecule positive granulocyte and lymphocyte cells (CD11a, CD11b, CD18 and CD49b) were measured on days 0, 7 and 14. Percentage of positive lymphocytes for CD54, CD49b, CD49d and CD8 were also measured on days 0, 7 and 14. The state of pressure ulcers was assessed by using the pressure ulcer scale for healing tool score on days 7, 14 and 28 of treatment. No between-group differences in patient demographics, anthropometry or diagnostic class were observed. Patients who received the study formula showed significant increases in the percentage of positive CD18 and CD11a lymphocytes and of CD49b granulocytes as compared to controls (P < 0·05). While the severity of pressure ulcers was not significantly different between the two groups on admission, severity increased significantly over time for the control group (P < 0·05), but not for the study group. The present study suggests that a fish oil- and micronutrient-enriched formula may prevent worsening of pressure ulcers and that this effect may be mediated by an effect on adhesion molecule expression.

Dual-functional capability of CD3+CD56+ CIK cells, a T-cell subset that acquires NK function and retains TCR-mediated specific cytotoxicity

AbstractCD3+CD56+ cytokine-induced killer (CIK) cells display a potent cytolytic activity. The adhesion molecule lymphocyte function-associated antigen-1 plays a crucial role in binding as well as in cytolytic activity of CIK cells against tumor target cells expressing the corresponding ligands. CIK cells express activating natural killer (NK) receptors, including NKG2D, DNAX accessory molecule-1 (DNAM-1), and low levels of NKp30. Cell signaling not only through TCR/CD3 but also through NKG2D, DNAM-1, and NKp30 leads to CIK cell activation resulting in granule exocytosis, cytokine secretion, and cytotoxicity. Antibody blocking experiments showed that DNAM-1, NKG2D, and NKp30 are involved in the TCR-independent tumor cell recognition and killing. Anti–CMV-specific CIK cells could be expanded in standard CIK cultures and mediate both specific, MHC-restricted recognition and TCR-independent NK-like cytolytic activity against leukemic cell lines or fresh leukemic blasts. Antibody blocking of lymphocyte function-associated antigen-1 and DNAM-1 led to significant reduction of both CTL and NK-cell functions, whereas blocking of NKG2D and NKp30 only inhibited NK-like cytotoxicity. Their dual-effector function suggests that CIK cells, when used in a clinical setting, may control both neoplastic relapses and viral infections, 2 frequently associated complications in patients who received a transplant.

Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity

As an approved vaccine adjuvant for use in humans, alum has vast health implications, but, as it is a crystal, questions remain regarding its mechanism. Furthermore, little is known about the target cells, receptors, and signaling pathways engaged by alum. Here we report that, independent of inflammasome and membrane proteins, alum binds dendritic cell (DC) plasma membrane lipids with substantial force. Subsequent lipid sorting activates an abortive phagocytic response that leads to antigen uptake. Such activated DCs, without further association with alum, show high affinity and stable binding with CD4(+) T cells via the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1). We propose that alum triggers DC responses by altering membrane lipid structures. This study therefore suggests an unexpected mechanism for how this crystalline structure interacts with the immune system and how the DC plasma membrane may behave as a general sensor for solid structures.

Porcine Sialoadhesin (CD169/Siglec-1) Is an Endocytic Receptor that Allows Targeted Delivery of Toxins and Antigens to Macrophages

Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab')2 fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages.

Decrease in antibody somatic hypermutation frequency under extreme, extended spaceflight conditions

Somatic hypermutation diversifies antibody binding sites by introducing point mutations in the variable domains of rearranged immunoglobulin genes. In this study, we analyzed somatic hypermutation in variable heavy-chain (VH) domains of specific IgM antibodies of the urodele amphibian Pleurodeles waltl, immunized either on Earth or onboard the Mir space station. To detect somatic hypermutation, we aligned the variable domains of IgM heavy-chain transcripts with the corresponding VH gene. We also quantified NF-κB and activation-induced cytidine deaminase transcripts. Results were compared with those obtained using control animals immunized on Earth. Our data show that, as in most species of ectotherms, somatic hypermutation in P. waltl exhibits a mutational bias toward G and C bases. Furthermore, we show for the first time that somatic hypermutation occurs in space following immunization but at a lower frequency. This decrease is not due to a decrease in food intake or of the B-cell receptor/antigen interaction or to the absence of the germinal center-associated nuclear protein. It likely results from the combination of several spaceflight-associated changes, such as the severe reduction in T-cell activation, important perturbations of the cytoskeleton, and changes in the distribution of lymphocyte subpopulations and adhesion molecule expression.

LFA-1 fine-tuning by cathepsin X

The adhesion molecule lymphocyte function-associated antigen (LFA)-1 plays a key role in immune surveillance and response. Its conformation is spatially and temporally regulated, enabling adhesion and deadhesion during T-cell migration. LFA-1 adhesion to its major ligand intercellular adhesion molecule 1 is controlled by adaptor proteins which bind the cytoplasmic tail of the β (2) subunit. Cathepsin X, a cysteine carboxypeptidase, promotes T-cell migration and morphological changes by cleaving the β (2) cytoplasmic tail of LFA-1. In this way, it modulates the affinity of LFA-1 for structural adaptors talin-1 and α-actinin-1 and enables the stepwise transition between intermediate and high-affinity conformations of LFA-1, an event that is necessary for effective T-cell function. Cathepsin X regulation that would allow precise modulation of LFA-1 affinity has a great potential for anti-LFA-1 therapy.

LFA-1–specific therapy prolongs allograft survival in rhesus macaques

Outcomes in transplantation have been limited by suboptimal long-term graft survival and toxicities associated with current immunosuppressive approaches. T cell costimulation blockade has shown promise as an alternative strategy to avoid the side effects of conventional immunosuppressive therapies, but targeting CD28-mediated costimulation alone has proven insufficient to prevent graft rejection in primates. Donor-specific memory T (TM) cells have been implicated in costimulation blockade-resistant transplant rejection, due to their enhanced effector function and decreased reliance on costimulatory signaling. Thus, we have tested a potential strategy to overcome TM cell-driven rejection by targeting molecules preferentially expressed on these cells, such as the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1). Here, we show that short-term treatment (i.e., induction therapy) with the LFA-1-specific antibody TS-1/22 in combination with either basiliximab (an IL-2Rα-specific mAb) and sirolimus (a mammalian target of rapamycin inhibitor) or belatacept (a high-affinity variant of the CD28 costimulation-blocker CTLA4Ig) prolonged islet allograft survival in nonhuman primates relative to control treatments. Moreover, TS-1/22 masked LFA-1 on TM cells in vivo and inhibited the generation of alloproliferative and cytokine-producing effector T cells that expressed high levels of LFA-1 in vitro. These results support the use of LFA-1-specific induction therapy to neutralize costimulation blockade-resistant populations of T cells and further evaluation of LFA-1-specific therapeutics for use in transplantation.

Time-dependent changes in the expression of lymphocyte and monocyte cell adhesion molecules after meals of different composition

The objective of the present study was to compare the acute effect of meals of different composition on the expression of adhesion molecules that play a key role in leucocyte trafficking. A total of twenty apparently healthy subjects randomly consumed three isoenergetic meals 1 week apart: enriched in carbohydrates (CHO), enriched in monounsaturated fat and enriched in saturated fat. Blood samples were obtained before the meals and at 2, 4, 6, 8 and 10 h after meal ingestion. Samples were analysed for LDL resistance to Cu-mediated oxidation and for the surface expression on peripheral blood mononuclear cells (PBMC) of CD62L, CD162, CD11a, CD11b, CD49d and CD54 by flow cytometry. The present results showed that there were no changes in LDL susceptibility to oxidation within and among the meals. After the CHO-enriched meal, there was a time-dependent increased expression of CD162, CD49d, CD11a and CD54 on PBMC that returned to basal values after 8-10 h. These changes were significantly greater than the ones observed after the consumption of the monounsaturated fat- and the saturated fat-enriched meals and were more evident in lymphocytes than in monocytes. In conclusion, acute ingestion of a CHO-enriched meal induces higher increases of lymphocyte activation markers than fat-enriched meals. These results suggest that long-term consumption of CHO-enriched diets may be associated with a sustained pro-inflammatory state.

Photochemotherapy for systemic sclerosis: effect on clinical and molecular markers

The cutaneous changes seen in systemic sclerosis (SSc) can result in considerable patient morbidity. We previously reported on the beneficial effect of psoralen ultraviolet A (PUVA) phototherapy in 13 patients with morphoea. We now report the findings of a study in which patients with SSc were treated with PUVA. Twelve patients with SSc were treated with PUVA phototherapy. The effect on cutaneous disease activity was assessed using the modified Rodnan score, and the effect on serological and immunohistochemical growth factors and adhesion molecules was also measured. The median Rodnan score at baseline was 24.5 [interquartile range (IQR) 18.5-26.0]. The median number of treatments with PUVA was 24 exposures (IQR 20-26) with a median cumulative exposure of 68.3 J/cm(2) (IQR 28.6-139.8). Of the 12 patients, 11 responded well to phototherapy with a mean change in Rodnan score of 6.58 (36.98%) (P < 0.01, Wilcoxon signed ranks test). After treatment with PUVA there was a significant increase in circulating tumour necrosis factor-alpha levels in 8/12 patients (P = 0.03). In 7/12 patients there was an increase in E-selectin and vascular cell adhesion molecule, although this was not significant. PUVA treatment is associated with a significant improvement in cutaneous symptoms in patients with SSc as measured by the Rodnan score (P < 0.01). Specific lymphocyte markers, adhesion molecules and cytokines are also affected by this treatment, helping to clarify further the mechanism of action of PUVA treatment and our understanding of the primary pathological process.

Expression of endothelia and lymphocyte adhesion molecules in bronchus-associated lymphoid tissue (BALT) in adult human lung

BackgroundBronchus-associated lymphoid tissue (BALT) is the secondary lymphoid tissue in bronchial mucosa and is involved in the development of bronchopulmonary immune responses. Although migration of lymphocytes from blood vessels into secondary lymphoid tissues is critical for the development of appropriate adaptive immunity, the endothelia and lymphocyte adhesion molecules that recruit specific subsets of lymphocytes into human BALT are not known. The aim of this study was to determine which adhesion molecules are expressed on lymphocytes and high endothelial venules (HEVs) in human BALT.MethodsWe immunostained frozen sections of BALT from lobectomy specimens from 17 patients with lung carcinoma with a panel of monoclonal antibodies to endothelia and lymphocyte adhesion molecules.ResultsSections of BALT showed B cell follicles surrounded by T cells. Most BALT CD4+ T cells had a CD45RO+ memory phenotype. Almost all BALT B cells expressed α4 integrin and L-selectin. In contrast, 43% of BALT T cells expressed α4 integrin and 20% of BALT T cells expressed L-selectin. Almost all BALT lymphocytes expressed LFA-1. HEVs, which support the migration of lymphocytes from the bloodstream into secondary lymphoid tissues, were prominent in BALT. All HEVs expressed peripheral node addressin, most HEVs expressed vascular cell adhesion molecule-1, and no HEVs expressed mucosal addressin cell adhesion molecule-1.ConclusionHuman BALT expresses endothelia and lymphocyte adhesion molecules that may be important in recruiting naive and memory/effector lymphocytes to BALT during protective and pathologic bronchopulmonary immune responses.

Prostaglandin E2Inhibits Advanced Glycation End Product-Induced Adhesion Molecule Expression, Cytokine Production, and Lymphocyte Proliferation in Human Peripheral Blood Mononuclear Cells

Advanced glycation end product (AGE) subtypes, proteins or lipids that become glycated after exposure to sugars, induce complications in diabetes. Among the various AGE subtypes, glyceraldehyde-derived AGE (AGE-2) and glycolaldehyde-derived AGE (AGE-3) have been indicated to play roles in inflammation in diabetic patients. The engagement of AGEs and receptor for AGEs activates monocytes. Because the engagement of intercellular adhesion molecule-1 (ICAM-1), B7.1, B7.2, and CD40 on monocytes with their ligands on T cells plays roles in cytokine production, we investigated the effects of AGE-2 and AGE-3 on the expressions of ICAM-1, B7.1, B7.2, and CD40 on monocytes, the production of interferon gamma and tumor necrosis factor alpha, and the lymphocyte proliferation in human peripheral blood mononuclear cells and their modulation by prostaglandin E(2) (PGE(2)). AGE-2 and AGE-3 induced the expressions of adhesion molecule, the cytokine production, and the lymphocyte proliferation. PGE(2) concentration-dependently inhibited the actions of AGE-2 and AGE-3. The effects of PGE(2) were mimicked by an E-prostanoid (EP)(2)-receptor agonist, 11,15-O-dimethyl prostaglandin E(2) (ONO-AE1-259-01), and an EP(4) receptor agonist, 16-(3-methoxymethyl)phenyl-omega-tetranor-3,7-dithia prostaglandin E(1) (ONO-AE1-329). An EP(2)-receptor antagonist, 6-isopropoxy-9-oxaxanthene-2-carboxylic acid (AH6809), and an EP(4)-receptor antagonist, (4Z)-7-[(rel-1S,2S,5R)-5-(1,1'-biphenyl-4-yl)methoxy)-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid (AH23848), inhibited the actions of PGE(2). The stimulation of EP(2) and EP(4) receptors is reported to increase cAMP levels. The effects of PGE(2) were reversed by a protein kinase A (PKA) inhibitor, H89, and mimicked by a dibutyryl cAMP and an adenylate cyclase activator, forskolin. These results as a whole indicated that PGE(2) inhibited the actions of AGE-2 and AGE-3 via EP(2)/EP(4) receptors and the cAMP/PKA pathway.

Increased percentage of L-selectin+ and ICAM-1+ peripheral blood CD4+/CD8+ T cells in active Graves' ophthalmopathy.

The purpose of the study was to evaluate the percentage of CD4+/CD8+ peripheral T cells expressing CD62L+ and CD54+ in patients with Graves' disease and to assess if these estimations could be helpful as markers of active ophthalmopathy. The study was carried out in 25 patients with Graves' disease (GD) divided into 3 groups: 1/ 8 patients with active Graves' ophthalmopathy (GO) (CAS 3-6, GO complaints pound 1 year), 2/ 9 patients with hyperthyroid GD without symptoms of ophthalmopathy (GDtox) and 3/ 8 patients with euthyroid GD with no GO symptoms (GDeu). The control group consisted of 15 healthy volunteers age and sex matched to groups 1-3. The expression of lymphocyte adhesion molecules was evaluated by using three-color flow cytometry. In GO group the percentage of CD8+CD54+, CD8+CD62L+, CD4+CD54+ and CD4+CD62L+ T cells was significantly higher as compared to controls (p<0.001, p<0.05, p<0.01, p<0.001 respectively). The percentage of CD8+CD54+ T lymphocytes was also elevated in GO group in comparison to hyperthyroid GD patients (p< 0.05). CD4+CD62L+ and CD8+CD54+ percentages were also increased in GDtox and GDeu as compared to controls. We found a positive correlation between the TSHRab concentration and the percentage of CD8+CD62L+ T cells in all studied groups (r= 0.39, p<0.05) and between the TSHRab level and CAS (r= 0.77, p<0.05). The increased percentage of CD8+CD54+ and CD8+CD62L+ T cells in patients with Graves' ophthalmopathy may be used as a marker of immune inflammation activity.

Effects on adhesion molecule expression and lymphocytes in the bovine mammary gland following intra-mammary immunisation

Changes to adhesion molecule expression and lymphocyte populations were evaluated in alveolar mammary tissue collected from cows following an immunisation protocol that involved intra-mammary inoculation to induce an IgA response in mammary secretions. The right quarters of the udder were immunised; the left side acted as a control. Antibody titres in secretions showed that at least two animals responded with antigen-specific IgA. Numbers of T-lymphocytes were 4-fold higher in immunised glands compared with controls ( P < 0.05). IgA-, IgM- and IgG-positive cell numbers were significantly higher ( P < 0.01) in immunised glands compared with controls in three of the four cows. No mucosal addressin molecule-1 (MAdCAM-1), vascular cell-adhesion molecule-1 (VCAM-1) or peripheral node addressin (PNAd) protein expression was detected on smaller venules that stained positively for von Willebrand factor in alveolar mammary tissues, from either immunised or control glands. Both VCAM-1 and PNAd were detected on smaller venules in supramammary lymph nodes, however, there was no significant difference between immunised and control glands. Quantification of MAdCAM-1 mRNA showed very low expression in both immunised and control alveolar tissue compared with Peyer's patch positive-control tissue. These findings suggest that the bovine mammary gland is capable of a mucosal antibody response; however, MAdCAM-1 is not involved with lymphocyte homing to the mammary gland in this species.

Photochemotherapy for localized morphoea: effect on clinical and molecular markers

Effective treatment options for morphoea remain limited. As a result, there has been increasing interest in the role of phototherapy in the management of this condition. Aims. We report the findings of a study in which 13 patients with localized morphoea were treated with oral (n = 11) and topical (n = 2) psoralen ultraviolet (PUVA) phototherapy. The clinical effect on disease activity was assessed using a skin score adapted from the modified Rodnan score. The effect on serological and immunohistochemical markers was also measured. In total, 11/13 patients showed an improvement in their skin score after phototherapy, with the mean reduction in score being 62.9% (P = 0.003, Wilcoxon signed rank test). After treatment with PUVA, there was a fall in circulating levels of vascular cell adhesion molecule in 10/13 patients (P = 0.059) and a significant increase in tumour necrosis factor-alpha in 9 of 13 patients (P = 0.036). In the five patients in whom CD3 and CD4 was measured, all showed a reduction in CD3 (P = 0.025), with a fall in CD4 (P = 0.046) seen in four of the five patients. PUVA is associated with clinical improvement in patients with morphoea, as shown by significant improvement in the skin score. However, in one patient who had been simultaneously started on ciclosporin, this improvement could not be attributed to the phototherapy alone. Although the sample size was small, we also found that certain lymphocyte markers, adhesion molecules and cytokines are affected by this treatment, helping to further clarify the mechanism of action of PUVA treatment.

The ITGB2 immunomodulatory gene (CD18), enterocolitis, and Hirschsprung's disease

Hirschsprung's disease (HSCR)–associated enterocolitis (HAEC) remains a major contributor to morbidity and mortality associated with HSCR, being sometimes difficult to diagnose in its subclinical form. Its pathogenesis appears to include impaired local defense mechanisms as well as dysfunctional immune response and leukocyte function. In this context, the ITGB2 (CD18) immunomodulation-related gene is a possible candidate in HAEC pathogenesis as it codes for the β-subunit of leukocyte adhesion molecule lymphocyte function-associated antigen 1, which has an established role in T-cell development and function. ITGB2/CD18 has also been linked to chronic colitis in both human and animal models involving defense mechanisms within colonic mucosa. There is therefore a fairly compelling case for the potential involvement of the ITGB2 (CD18) in HAEC pathogenesis. Aim The aim of this study was to investigate the ITGB2 immunomodulatory gene (CD18) in a cohort of patients with HSCR and explore its correlation with enterocolitis. Patients and methods Screening for mutations of the ITGB2 (CD18) gene was performed on DNA extracted from colonic tissue samples and whole blood of 33 HSCR patients controlled by analysis of 60 unaffected individuals from the diverse South African population. Polymerase chain reaction amplification was performed, followed by heteroduplex single-strand conformation polymorphism analysis and bidirectional semiautomated DNA sequencing analysis. Results Heteroduplex single-strand conformation polymorphism banding patterns of the ITGB2 gene showed variations in 22 HSCR patients (66%), 13 of whom had severe episodes of HAEC, and 6 others had milder symptoms. Of the 13, 6 (46%) had Down's syndrome–associated HSCR. Genetic variations included 1 mutation (D77N), 2 known (V367, V441), and 4 novel polymorphisms (−111T/C, 24G/T, 295G/A, 892A/G). Significant associations were identified in the exon 5′ untranslated promotor region ( P < .0001), exon 10 ( P < .0007), and the 3′ untranslated promotor region at 122G/A ( P < .0001) and 370 G/T positions ( P = .04). Those regions of the gene most frequently associated with HAEC and severe symptoms were those with more than 1 variant identified in the gene. Conclusions This study shows that impaired CD18 leukocyte and T regulatory cell regulation can probably be linked to a genetic ( ITGB2) predisposition to HAEC. It furthermore provides a possible genetic link to HAEC patient selection, identifying a potential molecular target.

Dimethylfumarate Reduces Leukocyte Rolling in Vivo through Modulation of Adhesion Molecule Expression

Esters of fumaric acid have a long tradition in the treatment of psoriasis. Dimethylfumarate (DMF) is perceived as the main active substance. However, the molecular mechanisms of DMF action are not completely understood. Here, we investigate the effects of DMF on lymphocyte adhesion molecule expression in vitro and interactions with endothelial cells in vivo. DMF dose-dependently reduced superantigen-induced expression of CD25, human leukocyte antigen-DR, and cutaneous lymphocyte antigen by 27, 22, and 48% on CD3-positive cells, respectively. No change was observed for CD54, VLA-4, and P-selectin glycoprotein ligand-1. An enhancement of CD69 expression was noted (22%). DMF led to a significant reduction in binding of human peripheral blood mononuclear cells (PBMCs) to E-selectin (72%), P-selectin (36%), and vascular cell adhesion molecule-1 (33%) in vitro. Intravital microscopy of PBMCs in ear vasculature of wild-type and knockout mice showed that rolling was mainly P-selectin-dependent and could be reduced by 61% through DMF incubation. We provide early evidence that DMF affects adhesion molecule expression on human leukocytes and their rolling behavior in vivo, indicating that DMF directly affects the initial step of leukocyte extravasation.

Anti–LFA-1 Monotherapy Prevents Neointimal Formation in a Murine Model of Transplant Intimal Hyperplasia

Cardiac allograft vasculopathy (CAV) is the pre-eminent cause of late cardiac allograft failure. It is characterized by a concentric intimal hyperplasia, which we designate transplant intimal hyperplasia (TIH). To date, blockade of the adhesion molecule lymphocyte function-associated antigen-1 (LFA-1) has been shown to be effective in preventing TIH in experimental models of transplantation, but only when combined with other immunosuppressants. In this study we explored the impact of monotherapy against LFA-1 in a carotid artery allograft model of TIH. B10A(2R) (H-2(h2)) mice were used as donors and C57BL/6 (H-2(b)) mice used as recipients. The recipients were treated with a monoclonal antibody against LFA-1alpha (M17/4) or isotype-matched control immunoglobulin. Grafts were harvested after 35 days and analyzed by histomorphometry and immunohistochemistry. Blood samples were taken and analyzed by differential cell count and alloantibody levels. We found that treatment with M17/4 resulted in a significant reduction in TIH compared with controls. Immunostaining revealed that LFA-1alpha blockade inhibited CD45+ leukocyte infiltration, prevented intimal smooth muscle cell (SMC) proliferation, and preserved the medial SMC population. Finally, we demonstrated a reduction in the serum alloantibody titer in the group treated with anti-LFA-1alpha when compared with controls. We have demonstrated for the first time that LFA-1alpha blockade on its own can prevent development of TIH in an experimental model. The concept of modulating LFA-1alpha-mediated leukocyte migration and T-cell activation may therefore be of relevance to clinical cardiac transplantation and, as such, represents a potential target for therapeutic intervention against clinical CAV.

Importance of IFN-γ-Mediated Expression of Endothelial VCAM-1 on Recruitment of CD8+T Cells into the Brain During Chronic Infection withToxoplasma gondii

Interferon-gamma (IFNgamma) is essential for preventing reactivation of chronic infection with Toxoplasma gondii in the brain. We examined the role of IFNgamma on lymphocyte and endothelial adhesion molecule expression and T cell recruitment into the brain during chronic infection with T. gondii in IFNgamma knockout (IFNgamma(-/-)) and wild-type (WT) mice. Although the number of cerebral vessels expressing intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) increased in both WT and IFNgamma(-/-) mice following infection, there were more VCAM-1(+) vessels in brains of infected WT than of infected IFNgamma(-/-) mice; in contrast, numbers of ICAM-1(+) vessels did not differ between strains. We did not detect endothelial E-selectin, P-selectin, MAdCAM-1, or PNAd in any of the brains. Significantly fewer CD8(+) T cells were recruited into brains of infected IFNgamma(-/-) than WT mice. Treatment of infected IFNgamma(-/-) mice with recombinant IFN-gamma restored the expression of VCAM-1 on their cerebral vessels and recruitment of CD8(+) T cells into their brains, confirming an importance of this cytokine for upregulation of VCAM-1 expression and CD8(+) T cell trafficking. In infected WT and IFNgamma(-/-) animals, almost all cerebral CD8(+) T cells were lymphocyte function-associated antigen-1 (LFA-1)(high), CD44(high), and CD62L(neg), and approximately 38% were alpha4beta1 integrin(+). In adoptive transfer of immune spleen cells, pretreatment of the cells with a monoclonal antibody (mAb) against alpha4 integrin markedly inhibited recruitment of CD8(+) T cells into the brain of chronically infected WT mice. These results indicate that IFN-gamma-induced expression of endothelial VCAM-1 and its binding to alpha4beta1 integrin on CD8(+) T cells is important for recruitment of the T cells into the brain during the chronic stage of T. gondii infection, although LFA-1/ICAM-1 interaction may also be involved in this process.