Characterization Of Cellular Infiltrate Research Articles

Abstract 385: Characterization of Cellular Infiltrate in Bioprosthetic Aortic and Mitral Valves

Background: Bioprosthetic xenogeneic valves tend to deteriorate over time, termed structural valve deterioration (SVD), leading to valvular dysfunction. Several studies have hypothesized that SVD is a result of a chronic immune-mediated rejection process; however, a paucity of data exists demonstrating this conclusively. Objectives: Demonstrate that valves explanted for SVD contain a significantly greater burden of immune cellular infiltrate compared to normal human heart valves. Methods: Adult patients undergoing bioprosthetic aortic or mitral valve explantation secondary to a diagnosis of SVD were included in this study. A control arm included valves explanted from hearts without valvular disease considered for transplantation that were not utilized. Specimens underwent immunohistochemical staining using standard techniques utilizing the following primary monoclonal antibodies: anti-CD68, anti-CD20, and anti-CD3. Results: Eighteen valves explanted for SVD were analyzed along with 4 control valves. All valves were explanted from male patients with a mean age of 62-years. Overall, 12% (n=1) of patients had diabetes mellitus, 50% (n=4) had hypertension, 12% (n=1) had renal dysfunction and 38% (n=3) were on a lipid-lowering medication. Our results demonstrate that valves developing SVD have a cellular infiltrate containing significantly more immune cells than normal human valves. Specifically, there are significantly more CD3+ T-cells (p<0.05) and CD68+ macrophages (p<0.05) present in the cellular infiltrate of the SVD valves compared to the normal controls. Furthermore, while the cellular infiltrate of the SVD valves contains CD20+ B-cells, plasma cells, and neutrophils, none of these cells are present on the normal control valves (all p-values <0.05). Conclusions: Our data suggest a significantly greater immune cell infiltrate can be found in explanted bioprosthetic valves developing SVD than normal human valves, providing support for the chronic-immune mediated rejection hypothesis. This study will ultimately lead to a better understanding of the immune mechanisms responsible for SVD in bioprosthetic xenograft valves and will contribute to our goal of developing novel therapeutic techniques to mitigate development of SVD.

Characterization of cellular infiltrate, detection of chemokine receptor CCR5 and interleukin-8 and RANTES chemokines in adult periodontitis.

Leukocyte migration is essential for immune surveillance of tissues by focusing immune cells to sites of antigenic challenge. The control of leukocyte migration depends on the combined actions of adhesion molecules and a vast array of chemokines and their receptors. The purpose of the present study was to investigate the involvement of Interleukin-8 (IL-8), RANTES, the associated infiltrating cells and expression of CCR5 chemokine receptors in periodontitis; furthermore, the effect of periodontal therapy on these parameters was evaluated. Patients included in the study had moderate to advanced periodontal disease with at least 5-6 teeth with probing depth > 6 mm, attachment loss > or =3 mm and extensive radiographic bone loss. The inflammatory infiltrate was analyzed by immunohistochemistry in gingival biopsies obtained from subjects at the beginning of the study and 2 months after periodontal treatment. Gingival crevicular fluid (GCF) was collected for 30 seconds using periopaper strips, and chemokines were quantified by ELISA. The cellular components of the inflammatory infiltrate included B (CD19) and T (CD3, CD4+ and CD8+) lymphocytes and monocytes/macrophages (CD11c). CCR5 chemokine receptor expressing cells were exclusively found in periodontitis gingiva. IL-8 and RANTES were detected in the periodontitis group, obtaining a total amount of 212.5 pg and 42.0 pg, respectively. However, IL-8 was also detectable in the GCF of the healthy group (total amount of 44.8 pg). Periodontal therapy reduced the cell number in the infiltrate and the levels of IL-8 and RANTES, suggesting a relationship between these chemokines and periodontal status. We propose that the presence of these chemokines and the expression of chemokine receptors may represent a marker of lymphocyte subsets with the ability to migrate to inflammatory sites.

Characterization of cellular infiltrates and cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response

Cryptococcosis, an increasingly important opportunistic infection caused by the encapsulated yeast-like organism Cryptococcus neoformans, is limited by an anticryptococcal cell-mediated immune (CMI) response. Gaining a thorough understanding of the complex anticryptococcal CMI response is essential for developing means of controlling infections with C. neoformans. The murine cryptococcosis model utilizing footpad swelling to cryptococcal antigen (delayed-type hypersensitivity [DTH]) has proven to be a valuable tool for studying the induction and regulation of the anticryptococcal CMI response, but this technique has limitations with regard to evaluating the role of the final effector cells recruited by an ongoing CMI response. The purpose of this study was to assess the types of cells and cytokines induced into the site of cryptococcal antigen deposition in C. neoformans-infected and -immunized mice compared with those for control mice. We used a gelatin sponge implant model to examine the cells and cytokines present at the site of an anticryptococcal DTH response. Sponges implanted in infected mice and injected with cryptococcal culture filtrate antigen (CneF) 24 h before assessment had significantly increased numbers of infiltrating leukocytes compared with saline-injected sponges in the same animals. Exaggerated influxes of neutrophils and mononuclear cells were the major contributors to the increase in total numbers of cells in the DTH-reactive sponges. The numbers of CD4+ and LFA-1+ cells were found to be significantly increased in the CneF-injected sponges of infected and immunized mice over the numbers in control sponges. The numbers of large granular lymphocytes were also increased in DTH-reactive sponges compared with control sponges. Gamma interferon, interleukin 2 (IL-2), and IL-5 are clearly relevant cytokines in the anticryptococcal CMI response, since they were produced in greater amounts in the CneF-injected sponges from C. neoformans-infected and -immunized mice than in control sponges. IL-4 was not associated with the expression of DTH to cryptococcal antigen. The gelatin sponge model is an excellent tool for studying cells and cytokines involved in specific CMI responses.

Characterization of cellular infiltrates in the rat urinary bladder following BCG and thiotepa intravesical therapy

We examined rat urinary bladders following intravesical administration of BCG and thiotepa. BCG administration resulted in a relatively greater increase in the mucosal infiltration of mononuclear cells relative to polymorphonuclear cells (P less than 0.01) compared to the thiotepa treated bladders. This finding suggests that the mode of action of the therapeutic effects of these agents may be different. These results may also suggest that the mechanism of action of BCG might be immunologic in nature.

CELLULAR PROLIFERATION AT THE SITE OF ORGAN ALLOGRAFTS AND THE INFLUENCE OF IMMUNOSUPPRESSIVE THERAPY

In this study we investigated cellular proliferation of T cells and macrophages at the site of rat cardiac allografts and determined the influence of immunosuppressive therapy on the proliferative characteristics of these cell types. A bromodeoxyuridine-labeling technique was used that allowed both the accurate detection of proliferative activity and the phenotypic characterization of cellular infiltrates within grafted tissues. In untreated recipients (BN----Lewis), T cytotoxic/suppressor cells as well as T helper cells showed proliferative activity at the site of the graft. The percentage of OX8-positive cells within the graft that showed proliferation ranged from 15% to 37%. The percentage of W3/25-positive cells within the graft that showed proliferation ranged from 25% to 30%. In contrast, macrophages hardly showed proliferative activity within the graft; only 1-4% of the macrophages stained positive for bromodeoxyuridine. From these observations it is concluded that the graft serves as a nonlymphoid tissue site, wherein lymphocytes can freely proliferate and expand. To study the influence of immunosuppressive therapy on cellular proliferation, the steroid budesonide, 120 micrograms/kg/day, was administered for 13 days (MST, 20 days). During effective immunosuppressive therapy, still a remarkable amount of infiltrating cells was present within the grafts. Moreover, immunosuppressive treatment did not primarily appear to affect the proliferative capacity of the individual cell types. OX8-positive cells as well as W3/25-positive cells clearly showed proliferative activity within the treated grafts. However, despite the presence of these proliferative cells, signs of graft destruction were absent during immunosuppressive therapy. This finding may shed new light on the effect of steroids at the site of the graft and their role in the prevention of tissue destruction.

Characterization and Quantification of Cellular Infiltrates in Nasal Mucosa of Patients with Grass Pollen Allergy, Non-Allergic Patients with Nasal Polyps and Controls

Little is known about cellular infiltrates in nasal mucosa and the differences between these infiltrates in allergic and non-allergic patients. A reproducible and objective method making use of monoclonal antibodies for the quantification and characterization of cellular infiltrates in biopsy specimens of nasal mucosa is described. This method was used to study quantitative differences in cellular infiltrates in the epithelium and lamina propria of the nasal mucosa of patients with isolated grass pollen allergy, non-allergic patients with nasal polyps, and controls. A surprisingly wide variation was found in all groups. In all groups the T lymphocytes were much more numerous than the B lymphocytes. The number of CD8+ cells exceeded the number of CD4+ cells in the epithelium but in the lamina propria the numbers were approximately equal. Significant differences between the three groups were found with respect to the number of CD1+, IgE+, neutrophils and cytoplasmic IgG4+ cells. No significant differences were found in the numbers of CD4+, CD8+, CD14+, CD22+, HLA-DR+, IgG1-3+ cells or eosinophils. The use of biopsy in combination with monoclonal antibodies is an easy and well-tolerated method to study immunological reactions in the nasal mucosa. The results of this study indicate a possible role for a T-cell-mediated response in allergic rhinitis.

Characterization of cellular infiltrates in skin lesions of atopic eczema by means of monoclonal antibodies.

Frozen skin biopsies from 3 acute and 3 chronic lesions of atopic dermatitis were examined by means of various ortho-monoclonal antibodies (OKT3, 4] not equal to----4, OKT6, OKT8; OKIa and OKM1) in an indirect immunofluorescence technique. The results show that both T helper (T4) and T suppressor cytotoxic (T8) lymphocytes are present in the inflammatory infiltration which predominantly contained Ia mononuclear cells. Despite the absence of double staining procedure, the observations suggest that T4 cells predominate over T8 cells in the acute lesions whereas the reverse tendency is noted in the chronic lesions. T8 cells are found mainly in the superficial dermis or even in the epidermis; by contrast, T4 cells tend to be more deeply located in the dermis. The lesions are characterized by a dramatic increase in the number of Langerhans cells (OKT6, OKIa+) that are found not only in the epidermis but also in the dermis.