Use of a polyvalent bacterial lysate in patients with recurrent respiratory tract infections: results of a prospective, placebo-controlled, randomized, double-blind study.

Respiratory tract infections (RTIs) are the most common infections in humans, and it is difficult to effectively treat patients with increased susceptibility to these ailments. LW 50020 (Luivac; Paspat oral), an oral immunomodulator consisting of the antigens of seven bacteria commonly involved in RTIs, has been developed for the induction of specific and nonspecific immune responses of the mucosa-associated lymphoid tissue. In this placebo-controlled study, the efficacy and safety of the tablet formulation of LW 50020 were evaluated in children and adults with recurrent RTIs. Tablets were taken once daily during two periods of 4 weeks each, interrupted by a treatment-free interval of 4 weeks. The main endpoint of the study, a clinical severity score that evaluated treatment benefits, was significantly lower in the second study period in patients treated with the bacterial lysate compared to patients given placebo. A comparison of the infection rates in the first and second study periods of patients treated with LW 50020 revealed a placebo-corrected reduction of 39% in children and a placebo-corrected reduction of 44% in adolescents and adults. The placebo-corrected duration of infections was shortened by 47% in children and by 55% in older patients. No serious drug-related side effects occurred. This study demonstrated that the oral bacterial immunomodulator LW 50020 is efficacious in treating patients with recurrent RTIs.

Anti-p53 antibodies in serum of smokers and head and neck cancer patients.

Inactivation of the p53 gene occurs in various tumors favouring the accumulation of genetic aberration. It is assumed that p53 protein can become immunogenic in stimulating the production of circulating anti p53 antibodies. Sera from 97 patients with primary and 30 patients with recurrent squamous cell carcinoma of the head and neck were examined for p53 autoantibodies with an enzyme-linked immunosorbent assay (ELISA). Sera from 42 patients with benign ENT-diseases and 28 healthy smokers served as the control. 38.1% (37/97) of the patients with primary and 36.6% (11/30) with recurrent SCCHN had autoantibodies to p53 in their serum. The evidence of p53 antibodies was not dependent on histological grading, T- or UICC stage of the disease. 24.2% (17/70) of the control group also had autoantibodies to p53 in the serum. The values of the control group and the patient group are so closely related, that p53 autoantibodies can not serve as a marker of malignancy. The indication for p53 measurement by ELISA should be very closely defined and limited.

Expression of ICAM-1 on intact cartilage and isolated chondrocytes.

A major factor in cellular cytotoxicity is the interaction between LFA-1 on leukocytes and ICAM-1 on targets. Because several inflammatory cartilage diseases are characterized by the presence of leukocyte infiltrates, the expression of ICAM-1 on human cartilage, cultured chondrocytes, and transplanted cartilage was investigated using monoclonal antibodies. Frozen tissue sections, chondrocytes in suspension, as well as total cellular mRNA were prepared from human cartilage samples. ICAM-1 expression was studied with two different monoclonal antibodies directed against ICAM-1 by immunohistochemical APAAP-staining and additional flow cytometric analyses. The expression of ICAM-1-mRNA in cartilage tissue was analyzed using the northern blot hybridization technique. Furthermore, chondrocytes were treated in culture with interleukin-1 (IL-1) and gamma-interferon (gamma-IFN). ICAM-1 expression after culture was quantified using flow cytometric analysis. We could detect ICAM-1 mRNA in cartilage tissue, however, the immunostaining of tissue sections using monoclonal antibodies did not give clear positive reactions. Isolated chondrocytes showed strongly positive staining patterns in comparison with adequate negative controls as assessed by flow cytometry. A dose-dependent increase of the expression of ICAM-1 on chondrocytes was observed when stimulated with IL-1 and gamma-IFN. Finally, two of the three studied transplanted autologous cartilage samples with advanced resorption showed the presence of ICAM-1 molecules as assessed by immunohistochemistry. This expression of ICAM-1 suggests that the molecule plays a role in severe cartilage inflammatory processes, where tissue damage leads to the exposure of chondrocyte surfaces.

Effect of growth factors on matrix synthesis by human nasal chondrocytes cultured in monolayer and in agar.

Reconstructive surgery of multiple areas of the body may require replacement bone or cartilage transplants to repair defects or lesions of skeletal tissue. Advances in cell and tissue culture techniques now permit synthesis of autologous human cartilage in vitro. Several growth factors regulate the metabolism and activation of cartilage cells. To enhance culture conditions and effectiveness for in vitro cartilage engineering, the aim of our investigations was to characterize the influence of transforming growth factor (TGF)-beta and basic fibroblast growth factor (bFGF) on human nasal septal chondrocytes. The isolated cells were cultured as monolayers on plastic and in soft agar. The biological effects of the growth factors were assessed by determining synthesis of total protein and proteoglycan. TGF-beta caused a dose-dependent stimulation of total protein as well as glycosaminoglycan synthesis by all chondrocytes cultured. This stimulatory effect of TGF-beta was greater for chondrocytes cultured in soft agar than for chondrocytes cultured on plastic. No stimulatory effects of matrix synthesis was observed for bFGF in either culture condition. Our results show that TGF-beta can be employed to enhance in vitro production of cartilage grafts for reconstructive surgery.

Expression of ICAM-1 on isolated human nasal, auricular and costal chondrocytes.

Expression of intercellular adhesion molecule-1 (ICAM-1) on targets has been reported to be a relevant factor for leukocyte migration, adhesion and function. Because stimulated chondrocytes have been shown to express molecules of immunological import (like HLA class II antigens) and because rejected or resorbed cartilage grafts used in the field of ENT are often characterized by adjacent infiltrating leukocytes, the presence of ICAM-1 on human nasal, auricular and costal cartilage was investigated. For this study, cartilage tissue sections and chondrocytes in suspension as well as cultured chondrocytes were prepared. Specific monoclonal antibodies (mAb) were used for immunocyto- and immunohistochemical Alkaline-Phosphatase-anti-Alkaline-Phosphatase staining (APAAP staining) as well as for flow cytometry analysis. ICAM-1 on healthy cartilage tissue sections was not found. On the other hand, both chondrocytes freed from matrix and cultured chondrocytes showed strongly positive staining patterns for ICAM-1. This result was obtained for chondrocytes from nasal, auricular as well as costal cartilage. This observed expression of ICAM-1 on chondrocytes with defective extracellular matrix demonstrates that cartilage cells are able to synthesize ICAM-1 without any paracrine stimulus from non-chondrocyte cells. It suggests that ICAM-1 plays a role in processes where tissue damage leads to the exposure of chondrocyte surfaces. Therefore, ICAM-1 expression on chondrocytes may also be a factor in destructive cartilage graft resorption.

Synthesis of human cartilage using organotypic cell culture.

The limited supply of fresh autologous cartilage tissue for use in reconstructive surgery necessitates the use of vital banked allografts. A feasible in vitro production of cartilage tissue composed of living cells requires the use of modern tissue culture techniques retaining the phenotypic characteristics of chondrocytes. With this purpose in mind, human chondrocytes were isolated and cultured using different culture procedures: monolayer, suspension and agar gel. The differentiation state of chondrocytes as well as proteoglycan and collagen syntheses were assessed by histochemical and immunohistochemical methods. Whereas chondrocytes in monolayer displayed an unstable phenotype and tended to dedifferentiate, in three-dimensional culture the chondrocytes remained morphologically, phenotypically and functionally differentiated. Furthermore, an accumulation of matrix products pericellularly was observed in the agar gel. The results suggest that three-dimensional cultures in agar gel may allow the in vitro production of bioartificial cartilage for transplantation.

[Cartilage transplantation in the area of the head-neck: comparative study of HLA class II antigen induction to chondrocytes in various culture systems]. / Knorpeltransplantation im Kopf-Hals-Bereich: vergleichende Untersuchung zur HLA-Klasse-II-Antigen Induktion auf Chondrozyten in verschiedenen Kultursystemen.

Autologous and homologous cartilage grafts are often used in reconstructive head and neck surgery. Unfortunately, sometimes the outcome of such operations is endangered by graft rejection or resorption. Among other reasons, immunological reactions with HLA class II antigen expression are thought to be involved at least in failures of vitally grafted cartilage. Up to now, only one case of class II antigen expression in a cartilage graft "in vivo" has been reported. Nevertheless, it has already been demonstrated that stimulated cartilage cells are able to express "in vitro" class II antigens if grown in monolayer cultures. However, it has also repeatedly been shown that chondrocytes reveal strong dedifferentiating features if cultured in monolayers. Therefore, it was the aim of this study to examine whether isolated and stimulated chondrocytes also express class II antigens if cultured under in vitro conditions closer to the relevant in vivo situation. Hence monolayer, suspension, agar, alginate and organ cultures were prepared simultaneously and kept for up to 60 days. Chondrocytes were stimulated by the addition of IFN-gamma and tested for class II antigens. For the detection of the antigens immunocyto- and immunohistochemical APAAP stainings as well as flow cytometric measurements were made. In all examined culture systems a class II antigen induction could be observed. Any significant differences between the various cultures as to the intensity of antigen expression could not be detected. Consequently, the expression of class II antigens on stimulated human chondrocytes seems not to be a specificity of monolayer cultures. Therefore, class II antigen induction may be considered to play a role in the rejection/resorption of vitally grafted cartilage in reconstructive surgery.

Aberrant expression of epidermal growth factor receptor in aural cholesteatoma.

A monoclonal antibody recognizing an epitope of the external domain of the human epidermal growth factor (EGF) receptor was used in an alkaline phosphatase-antialkaline phosphatase (APAAP) technique to compare the distribution of this protein in normal human skin and aural cholesteatoma. EGF receptors appear to be highly expressed on the basal layer of the epidermis, in hair follicle apocrine sweat glands, and in the capillary system of normal skin. Cholesteatoma epithelium showed increased positive reactions in the suprabasal layers. A heterogeneity in the expression was found in different parts of the cholesteatoma. These results suggest the presence of an aberrant regulation and persistence of EGF receptors in cholesteatoma and confirm the hyperproliferative character of the cholesteatoma epithelium.

A critical analysis of human immunodeficiency virus transmission using human cartilage allografts.

Allogeneic cartilage represents an important source of tissue for reconstructive surgery in the head and neck. The use of allografts is now being discussed because of the possible transmission of the human immunodeficiency virus (HIV). The receptor for HIV in most cell types is the CD-4 molecule. Since cartilage is a popular homograft source, the purpose of this study was to investigate the presence of CD-4 molecules on cartilage tissue as detected with an immunoperoxidase staining and immunofluorescence flow cytometric analysis using a monoclonal antibody. Our results indicate clearly the absence of the HIV receptor on human cartilage tissue. We have concluded therefore that normal cartilage tissue cannot be infected by HIV, at least not through a CD-4-dependent mechanism.

Culture and cryopreservation of chondrocytes from human cartilage: relevance for cartilage allografting in otolaryngology.

One of the reasons for failure of cartilage allografts is the impaired condition of the transplant during storage. In this paper we describe methods for the isolation and culture of viable chondrocytes obtained from nasal septum cartilage. Furthermore, we evaluate the possibility of growing such specific chondrocytes under culture conditions and storing them in a frozen state. Age-dependent differences were observed in the growth rate of the cultured cells. Our results confirm that chondrocytes survive freezing and remain able to proliferate. Knowledge gained from this study may be applied to the culture and freezing of viable intact cartilage for use in reconstructive surgery in otolaryngology.