Andrew I. Lazarovits

Differential expression of tissue-specific adhesion molecules on human circulating antibody-forming cells after systemic, enteric, and nasal immunizations. A molecular basis for the compartmentalization of effector B cell responses.

Expression of the adhesion molecules CD44, L-selectin (CD62L), and integrin alpha 4 beta 7 by antibody-secreting cells (ASC) was examined in human volunteers after oral, rectal, intranasal, or systemic immunization with cholera toxin B subunit. Almost all blood ASC, irrespective of immunization route, isotype (IgG and IgA), and immunogen, expressed CD44. On the other hand, marked differences were observed between systemically and intestinally induced ASC with respect to expression of integrin alpha 4 beta 7 and L-selectin, adhesion molecules conferring tissue specificity for mucosal tissues and peripheral lymph nodes, respectively. Thus, most ASC induced at systemic sites expressed L-selectin, whereas only a smaller proportion of ASC expressed alpha 4 beta 7. In contrast, virtually all IgA- and even IgG-ASC detected after peroral and rectal immunizations expressed alpha 4 beta 7, with only a minor fraction of these cells expressing L-selectin. Circulating ASC induced by intranasal immunization displayed a more promiscuous pattern of adhesion molecules, with a large majority of ASC coexpressing L-selectin and alpha 4 beta 7. These results demonstrate that circulating ASC induced by mucosal and systemic immunization express different sets of adhesion molecules. Furthermore, these findings provide for the first time evidence for differential expression of adhesion molecules on circulating ASC originating from different mucosal sites. Collectively, these results may explain the anatomical division of mucosal and systemic immune responses in humans as well as the compartmentalization of mucosal immune responses initiated in the upper vs. the lower aerodigestive tract.

Pattern of liver, kidney, heart, and intestine allograft rejection in different mouse strain combinations.

With advances in microsurgery and molecular biology, the mouse model for organ transplantation has become increasingly popular. However, knowledge about these models is limited, as only a small number of centers have experience with murine models. In this study, we compared the rejection pattern after liver, kidney, heart, and small bowel transplantation in the three different mouse strain combinations: (1) C57BL/6 (H2b)-->BALB/c (H2d), (2) BALB/c (H2d)-->CBA (H2k), and (3) C57BL/6-->C3H/HeN (H2k). Our study demonstrated that mouse allograft survival varies depending on the organ graft and on the donor-recipient strain combinations. The majority of liver allografts were spontaneously accepted despite complete MHC disparity. A mixed pattern of acute rejection and acceptance occurred in kidney recipients depending on the donor-recipient strain combination. All the heart grafts developed rejection and all the intestinal grafts were rapidly rejected with no spontaneous acceptance. The criteria for rejection, the potential applications, and the limitations of each model are discussed. The models described in this article provide a number of useful choices for organ transplantation research.

The Outcome Of Pregnancy Following Renal Transplantation—the Experience Of A Single Center

Many centers still recommend avoidance of pregnancy after renal transplantation because of fears for the safety of both mother and fetus. These fears are in part based on a lack of information concerning the effects of newer immunosuppressive drugs such as cyclosporine on the course and outcome of pregnancy. The present study examines the experience of first pregnancies following renal transplantation in a single center, with emphasis on the role of CsA. Data on the first pregnancies of 22 women transplanted between 1977 and 1988 were studied. The mean age of patients at the time of transplant was 23.4±3.1 years and interval from transplant to pregnancy was 34.5±24.5 months (range 1–75 months). Twelve patients received CsA alone or in combination with other immunosuppressives, while the remaining 10 patients received azathioprine and prednisone. Mean serum creatinine fell progressively during pregnancy in both CsA-and azathioprine-treated mothers. Mean CsA dose rose during pregnancy while mean CsA blood concentration fell during the 2nd trimester (P=0.042). The gestation period ranged from 27 to 40 weeks (35.5±3.3) with 14 pregnancies ending prematurely prior to 37 weeks. Thirteen deliveries occurred by Caesarian section. Hypertension complicated 10 pregnancies. Birth weight correlated directly with both maternal weight gain (r=0.57; P < 0.02) and gestational age (r=0.9; P < 0.01). Ten of 23 offspring were below the 10th percentile for weight. Mean birth weight ranged from 0.72 to 3.7 kg (2.3±0.84 kg). The mean birth weight and gestational age of children born to mothers taking CsA were lower than those in azathioprine treated mothers but these differences were not statistically significant. Successful pregnancy is possible following renal transplantation, although there is a high rate of prematurity, low birth weight, and intrauterine growth retardation. CsA dose requirements may be increased. Maternal risks including hypertension require that such pregnancies be handled by a multidisciplinary team approach.

Analysis of peripheral blood lymphocyte populations and immune function from children exposed to cyclosporine or to azathioprine in utero

We have analyzed PBL from 7 children exposed to CsA in utero and 4 children exposed to AZA in utero. Expression of CD3, CD4, CD8, and CD20 were normal for both groups of children; however, significant differences were detected in the expression of CD45RA, CD45R0, and CD29. While CsA-exposed children had higher density of CD45RA, and a higher proportion of CD45RA+R0- T cells, than did unexposed children, those exposed to AZA alone had decreased CD45RA+ and a large increase in CD45RA-R0+ T cells. It appears the exposure to CsA slightly delays T cell development, whereas exposure to AZA, without concomitant exposure to CsA, accelerates development to that of an adult. The effects of CsA abrogated the effects of AZA when both were present during pregnancy. The expression of CD29, the beta 1-integrin, on T cells has been linked to enhanced ability to respond to recall antigens and to home to sites of infection. Among children exposed to CsA, T cells from cord blood and a 5-month-old infant have a normal CD29 profile. However from 1 to 6 years of age the proportion of T cells expressing a high density of CD29 is significantly lower (4-fold) than that of T cells from unexposed children. Because these children have no outward signs of immunodeficiency, we postulate that this low proportion is still sufficient for normal immune responsiveness. The distribution of CD29 on T cells was different for the 3 study groups. Among CsA-exposed children, although the proportion of CD29hi T cells was much reduced, all were CD45RA+, as was also the case for unexposed children. In contrast, among children exposed only to AZA, the majority of CD29hi T cells were CD45R0+. Serological testing indicated that immunoglobulin and complement levels, as well as seroconversion in response to vaccination, were normal among CsA-exposed children, with no detectable autoantibodies to cellular or tissue components, including parietal cells. Unlike T cell development in inbred rodents, the immune system in humans appears to be remarkably resilient, and successfully adapts to the presence of CsA during its early developmental stages. This work suggests that the presence of CsA throughout pregnancy has only a minimal effect on fetal immune development and appears to have less impact on T cells than does exposure to AZA only. We conclude that children exposed to CsA in utero are not likely to be at risk of immunodeficiency or autoimmunity.

Crohn's disease, ulcerative colitis, and normal intestinal lymphocytes express integrins in dissimilar patterns

BACKGROUND/AIMS The integrin family of adhesion molecules on intestinal lamina propria mononuclear cells (LPMNC) was studied using fluorescence-activated cell cytometry. These molecules are implicated in extravascular cell migration and are important regulators of disease. METHODS Using fluorescence-activated cell cytometry, B- and T-cell subsets in the intestines of 10 normal patients, 11 patients with Crohns disease, and 8 patients with ulcerative colitis were stained with monoclonal antibodies to a panel of integrins. RESULTS Expression of alpha integrins on CD3+ T cells and CD19+ B cells was different in normal and inflammatory bowel disease LPMNC. Ulcerative colitis T cells expressed less beta 1 and alpha 4 and significantly more alpha 2 and alpha 6. There was a difference in alpha 4 and beta 1 expression between LPMNC B cells from Crohns disease and normal intestines. Sixteen percent of CD19+ LPMNC B cells from Crohns and 19% of ulcerative colitis LPMNC expressed alpha 2. Crohns and ulcerative colitis CD19+ LPMNC B cells expressed more alpha 5 integrin than normal specimens. CD3+ T cells and CD19+ B cells expressed alpha 6 only in ulcerative colitis. Ulcerative colitis and Crohns disease CD19+ LPMNC expressed less alpha 4, consistent with their reciprocal increases of alpha 5 and alpha 2. A difference in beta 7 (Peyers patch specific) antigen was observed between inflammatory bowel disease and normal LPMNC for both CD3+ and CD19+ LPMNC. CONCLUSIONS These findings identify the differences of lymphocyte homing capability in inflammatory bowel disease and normal intestine.

Indefinite islet allograft survival in mice after a short course of treatment with anti-CD45 monoclonal antibodies.

BACKGROUND Although islet cell transplantation is considered an ideal form of endocrine replacement for type I diabetes, clinical application in humans is still not feasible. New immunosuppressive strategies are clearly needed to control inexorable rejection. CD45 is a family of transmembrane protein tyrosine phosphatases critically involved in the regulation of lymphocyte activation signals. Anti-CD45RB monoclonal antibody can prevent rejection of murine renal allografts. METHODS Here, we examine the consequences of targeting CD45 in murine islet cell transplantation. Diabetic mice recipients received islet allografts under the kidney capsule and were divided into seven groups. Recipients received no treatment (controls) or anti-CD45RB monoclonal antibody (mAb; MB23G2 or C363.16A) at different dosages and treatment intervals. RESULTS All untreated control animals lost islet function, becoming hyperglycemic within 10-17 days after transplantation. Animals treated with either anti-CD45RB mAb showed a significant prolongation of islet allograft survival when compared with controls. Anti-CD45RB MB23G2 at 100 microg/day, given on days -1, 0, and 5 was particularly effective, inducing indefinite islet allograft survival in 60% of recipients. CONCLUSIONS These results indicate that anti-CD45 mAbs are potent immunomodulatory agents, able to sustain indefinite islet allograft function after a short treatment course in the highly immunogenic model of islet transplantation.

Expression of the mucosal homing receptor α4β7 in malignant lymphomatous polyposis of the intestine

Abstract Recent studies have identified the integrin α4β7 as a mucosal homing receptor that mediates lymphocyte migration to the intestinal mucosa by binding to MAdCAM-1, which is a vascular recognition molecule (adressin) selectively expressed on mucosal endothelium. The expression of the α4β7 mucosal homing receptor was studied in eight cases of malignant lymphomatous polyposis (MLP). This unusual presentation of non-Hodgkins lymphoma of mantle cell type is characterized by multifocal lymphomatous involvement of the gastrointestinal tract. Unlike nodal mantle cell lymphomas, cases of MLP showed expression of α4β7, suggesting that this homing receptor plays an important role in determining the characteristic mucosal dissemination pattern of MLP.

Integrin α4β7 Co-Stimulation of Human Peripheral Blood T Cell Proliferation

The Integrin α4β7 mediates lymphocyte adhesion to VCAM-1 on activated endothelium, fibronectin in the extracellular matrix, and the mucosal vascular addressin MAdCAM-1. It is unclear whether α4β7 performs any function beyond directing specific adhesion reactions. We addressed the possibility that triggering of α4β7 with a specific monoclonal antibody was capable of delivering an accessory stimulus that would coactivate T cells and lead to proliferation. At submitogenic levels of anti-CD3 stimulation, triggering of α4β7 by immobilized mAb ACT-1 resulted in T cell blastogenesis, IL-2 production, expression of the IL-2 receptor α chain CD25, and ultimately DNA synthesis. These results indicate that the integrin α4β7 is involved in more than lymphocyte adhesion and homing but also plays a role in cell signaling.

Inhibition of alloreactivity in vitro by monoclonal antibodies directed against restricted isoforms of the leukocyte-common antigen (CD45).

The leukocyte common antigen (LCA) is a protein tyrosine phosphatase and is identified by the CD45 cluster of monoclonal antibodies. CD45 is expressed in high-density on cells of hematopoietic lineage and generally is immunoprecipitated as 4 bands (220, 205, 190, and 180 KDa). Genetic studies have shown that a single gene produces additional forms of the molecule by alternate splicing including CD45RA (220, 205), CD45RO (180), and CD45RB (220, 205, 100). We have prepared a CD45RB Mab termed “MT3” that binds to a sialic acid dependent epitope. Since the LCA is one of the major targets of antilymphocyte globulin, we assessed a panel of CD45 and CD45R Mab for their ability to inhibit alloreactivity in vitro. MT3 (CD45RB) inhibits the allogeneic MLR and inhibits CD4* T cells from expressing interleukin 2 receptors, and prevents CD4* CD45RA“ T cells from entering the proliferative phase of the cell cycle. Mem 93 (CD45RA) inhibits the generation of cytotoxic T cells. These data suggest that the CD45RB and CD45RA iso-forms of the LCA may be appropriate targets for in vivo immunotherapy.

Prolongation of xenograft survival using monoclonal antibody CD45RB and cyclophosphamide in rat-to-mouse kidney and heart transplant models.

BACKGROUND Intrigued by the finding that a monoclonal antibody (mAb) directed against the B exon of restricted CD45 (CD45RB mAb) induced renal allograft tolerance in the mouse model, we hypothesized that CD45RB mAb may prevent xenograft rejection. We explored the role of CD45RB mAb in preventing xenograft rejection in rat-to-mouse kidney and heart transplant models. METHODS Mice with rat kidney and heart xenografts were treated with a short course of mAb, cyclosporine, cyclophosphamide, or mAb + cyclophosphamide combination therapy. Untreated heart and kidney xenografts served as controls. RESULTS Untreated controls developed acute vascular and cellular rejection rapidly with a median survival time of only 6 days. Long-term kidney (median survival time = 70 days) and heart xenograft survival (median survival time = 65 days) was achieved using the combination therapy of mAb + cyclophosphamide. One-third of the kidney recipients with combination therapy survived 100 days. Immunohistochemistry and xenospecific-antibody analysis demonstrated that combination therapy remarkably reduced IgG and IgM deposition and also inhibited CD4+, CD8+, and Mac-1+ cell infiltration at early stages. This therapy, however, did not induce tolerance in this model as evoked xenoreactive antibodies and cellular responses may be the cause of late xenograft failure. CONCLUSION A short course of CD45RB mAb combined with cyclophosphamide effectively inhibits cellular and humoral immunoresponses and remarkably prolongs xenograft survival in rat-to-mouse heart and kidney transplant models.