Chronic infections and histamine, CRP and IL-6 levels after percutaneous transluminal coronary angioplasty.

OBJECTIVE AND DESIGN: Our hypothesis was that percutaneous transluminal coronary angioplasty (PTCA) reactivates certain pathogens that contribute to inflammatory processes after the intervention. SUBJECTS: We determined the levels of antibodies to human Hsp60 and levels of histamine, CRP and IL-6 in sera from 28 patients of unstable angina prior to and on days 4 and 14 after PTCA. We compared the presence of Chlamydophila pneumoniae (Cpn) and human cytomegalovirus (HCMV) DNA in peripheral blood, and levels of antibodies to Cpn, HCMV, herpes simplex virus, Epstein-Barr virus and mycobacterial Hsp65 in the serum. RESULTS: Higher prevalence of Cpn and HCMV DNA was demonstrated after PTCA than before, but titers of antibodies to the pathogens did not increase. Levels of histamine, CRP and IL-6 were enhanced after PTCA. There was no association between the levels of histamine, CRP and IL-6 and the rate of pathogen DNA, or antibody titers to the pathogens, except an association between Cpn IgA and histamine levels before PTCA. CONCLUSIONS: Reactivation of Cpn and HCMV and inflammatory change characterized by increased levels of histamine, CRP and IL-6 following PTCA are suggested. An association might exist between Cpn IgA antibody and histamine levels in patients of unstable angina.

Endotoxins do not influence transplacental transmission of lymphotropic human herpesviruses and human papillomaviruses into amniotic fluid taken from healthy mothers before parturition in Hungary.

Pregnant women were examined following healthy pregnancies at term. Amniotic fluids were sampled before arteficial rupture of membranes using closed vacutainer system. Blood samples were also taken from the pregnants simultaneously. Endotoxin concentrations of amniotic fluids were tested by the semiquantitative Limulus amebocyte lysate. Both amniotic fluids and blood samples were tested for the presence of DNA of lymphotropic human herpesviruses. The DNA of human papillomaviruses were tested only in the amniotic fluid samples. One-third of the amniotic fluids tested were found to contain measurable amounts of endotoxin. Lymphotropic herpesvirus DNA was deteced in every fourth amniotic fluid sample and in every 8th blood sample. The prevalence of papillomaviruses was 7 of 96 samples. No significant correlation was found between the presence of endotoxin and viruses in the amniotic fluids. Epstein-Barr virus, human cytomegalovirus and human herpesvirus type 7 were found more frequently in the amniotic fluids than in blood samples (7 to 1). The prevalence of human herpesvirus 6 and 8 was higher in the blood samples than that in the amniotic fluids. The mean weight of the neonates were not impaired significantly by the presence of either viruses or endotoxin. Possible post partum consequences, i.e. partial immunotolerance to viruses is discussed.

The interactions between human dendritic cells and microbes; possible clinical applications of dendritic cells.

The dendritic cells comprise several subsets that induce and regulate the immune responses against foreign and self-antigens, and that can therefore function as initiators of protective immunity and inducers of central or peripheral tolerance. The different subpopulations of dendritic cells interact with and also influence other cell populations of the immune system, such as T and B lymphocytes and natural killer cells. The factors that determine the given dendritic cell functions depend on the state of maturation and the local microenvironment. The interactions between dendritic cells and microorganisms are rather complex, but progress in the past few years has shed light on several aspects of these interactions. This review lays emphasis on the interactions between human dendritic cells, important components of the intima of arterial specimens at areas predisposed to atherosclerotic lesions, and Chlamydia pneumoniae and cytomegalovirus, the human pathogens most strongly implicated in the development of atherosclerosis. In addition, several examples of the potential clinical applications of dendritic cells are described.

Chlamydia pneumoniae exacerbates aortic inflammatory foci caused by murine cytomegalovirus infection in normocholesterolemic mice.

Inflammatory foci induced by murine cytomegalovirus infection in normocholesterolemic mice were present temporarily in the aortic wall, but some of these foci developed into advanced lesions that persisted late after infection. The early foci induced by virus infection were significantly exacerbated following a single inoculation with Chlamydia pneumoniae.

[Role of thalidomide in the treatment of multiple myeloma]. / A thalidomid szerepe a myeloma multiplex gyógykezelésében.

Multiple myeloma is a relatively common hematologic malignancy with no definitive treatment available. Although, therapy may include allogenic bone marrow transplantation, high-dose ablative chemotherapy followed by bone marrow or peripheral stem cell transplantation, melphalan/corticosteroid therapy, alpha-interferon treatment, and combined cytostatic chemotherapy, currently none of these alternatives offers cure for the disease. Thalidomide is an infamous molecule for its teratogenicity, yet it possesses potent immunomodulatory, anti-angiogeneic and, in higher concentrations, direct anti-myeloma-cell properties. At present, the drug is only approved for the treatment of erythema nodosum of leprosy, however, there are several preliminary results that show clinical efficacy in multiple myeloma. This drug has especially potent anti-myeloma effects in combinations with dexamethasone and certain cytostatic chemotherapeutic agents. The effects are evident both in polyresistant, and relapsing myeloma, a form with no accepted effective treatment options. In this paper, the fundamental molecular and cellular effects of thalidomide are summarized then the most important clinical studies with thalidomide are reviewed. It is the authors' hope that thalidomide will soon be a full member of the medical arsenal in the fight against multiple myeloma.

Differential in situ distribution of interleukin-8, monocyte chemoattractant protein-1 and Rantes in human chronic periapical granuloma.

In situ distribution of three prototype chemokines interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1 and Rantes was determined in chronic human periapical granulomas by immunohistochemistry using monoclonal antibodies. IL-8 was found primarily in the cytoplasm of the Malassez epithelial cells. MCP-1 immunoreactivity was confined to the endothelial cells that lined small venules. Each of the three investigated chemokines, including Rantes, exhibited a characteristic binding pattern to the extracellular matrix of the lesion. The observed chemokines may play a role in establishing the cellular composition of chronic apical periodontitis, thus augmenting the intensity of local inflammation and tissue damage.

Human herpesvirus 8 in hematologic diseases.

Human herpesvirus type 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV) is a new member of the g-herpesvirus family. It is an unusual herpesvirus in that it carries a large number of genes that encode oncoproteins or cell signaling proteins. In addition to being the causative agent of both HIV-associated and non-HIV-associated Kaposi's sarcoma this DNA tumor virus has been implicated in the pathogenesis of several diseases. These include multiple myeloma (MM), Waldenstöm's macroglobulinemia (WM), multicentric Castleman's disease (MCD), body cavity-based lymphoma (BCBL), and various other conditions such as sarcoidosis and pemphigus. While the causative role of the viral infection is fairly certain in the development of BCBL and multicentric Castleman's disease, HHV-8 may act through a different mechanism to induce plasma cell malignancies. It has been suggested though the finding is still controversial - that infection of bone marrow stromal dendritic cells by HHV-8 might be a key factor in the etiology and pathogenesis of monoclonal gammopathies. The aim of this review is to provide a short introduction into the tumorigenic potential of HHV-8 as well as to detail the available data and possible mechanisms on the involvement of this virus in different hematologic diseases.

Application of near infrared spectroscopy to the determination of haemoglobin.

Seventy-two whole blood samples were investigated to determine the relationship between their spectral data measured in the near infrared (NIR) wavelength region and haemoglobin content based on laboratory data determined by a routine standard method as reference. Blood samples were obtained from the 1st Department of Medicine, Imre Haynal University of Health Sciences. Donors were selected randomly without respect to age, sex, state of health or medical treatment, from apparently healthy volunteers as well as from ambulatory and hospitalized patients. NIR spectra were measured with a SPECTRALYZER 1025 (PMC) computerized spectrophotometer in the 1000-2500 nm wavelength region. The relationship between laboratory data and values of the second derivative (i.e. second order finite difference) of the log(1/TF) spectra measured at different wavelengths was determined by multiple linear regression (MLR) using three- and four-term linear summation equations. The cross-validated standard error of performance (SEP) for haemoglobin was 1.348 g dL-1 with a three term model and 1.251 g dL-1 with a four term model over the range from 5.9 to 20 g dL-1. This preliminary study indicates that NIR measurements can be directly related to haemoglobin content and can be used to determine haemoglobin content in human whole blood.

Theoretic opinion and future treatment of amyloidosis.

Amyloidosis is still enigmatic. The etiology and mechanism of storage and organ impairment are still unknown. Not all amyloidogen molecules are definitely pathogenic; some precursors of tissue amyloid are structurally analogous to normal substances produced in the organism. This paper reviews the currently accepted categories, aspects of etiology and pathogenesis, and therapeutic trends of amyloidosis with a special emphasis on plasmapheresis.

Early region 3-replacement adenovirus recombinants are less pathogenic in cotton rats and mice than early region 3-deleted viruses.

BACKGROUND: Adenovirus type-5 (Ad5) recombinant viruses with replacement of the 1.9 kb XbaI fragment in the early region 3 (E3) by foreign genes have been constructed with the ultimate goal of inducing immune responses to the product of the inserted gene against a variety of virus infections. The pathogenicity of these recombinants, however, has not been studied. EXPERIMENTAL DESIGN: Histopathologic changes induced in cotton rat and mouse lung by E3-replacement-Ad5 recombinant or wild-type Ad (Wt-Ad) or E3-deleted mutant (Ad5-delta E3) viruses were compared. Expression of viral mRNA and replication of these viruses in cotton rat and mouse lungs, as well as in human tissue culture cells, were assayed. Expression of class I major histocompatibility complex antigens and the E3-14.7 kilodalton protein in virus-infected cells were also analyzed. RESULTS: An Ad5 recombinant, Ad-human cytomegalovirus glycoprotein B (Ad-HCMV.gB), in which the E3 region is replaced by the full-length gB gene of HCMV and with a genome size exceeding that of Wt-Ad, induced mild histopathologic responses in cotton rat and mouse lungs, comparable with those of Wt-Ad, but less severe than those of Ad5-delta E3. Analysis indicated that neither class I major histocompatibility complex expression on the cell surface nor differential expression of the protective E3-14.7 kilodalton protein underlies the pathologic differences observed in cells infected with Ad5-delta E3 or the Ad-HCMV.gB recombinant. In the mouse lung, another Ad-E3 replacement recombinant, Ad-herpes simplex glycoprotein B (HSV.gB), containing the complete HSV.gB gene and with a genome size larger than that of Wt-Ad, also induced a very mild inflammatory response. However, two recombinants with truncated forms of the HCMV.gB (Ad-HCMV.gB.155) or HSV.gB genes (Ad-HSV.gB.147) produced more severe histopathologic changes than the Wt-Ad or the recombinants with the full complement of HCMV.gB or HSV.gB genes. Ad5 and some of the recombinants replicated in mouse and cotton rat lung, and the extent of replication was inversely proportional to genome size, both in the lung and in human tissue culture cells. Infectious virus titers were, however, higher in cotton rat than in mouse lung. In situ hybridization analysis of cotton rat and mouse lung infected with Wt-Ad, Ad5-delta E3, or Ad-HCMV.gB virus revealed expression of Ad early/late mRNA predominantly in bronchial epithelial cells. CONCLUSIONS: These data not only confirm that E3-deleted viruses induce more severe pathologic changes in cotton rat lungs than Wt-Ad viruses (Ginsberg et al., Proc Natl Acad Sci USA 1989;86:3823-7) but led to the observation that some E3 replacement recombinants also lacking the expression of the 19 and 14.7 kilodalton proteins are significantly less pathogenic in cotton rats and mice than an E3-deleted virus. Pathogenicity and replication of the recombinant viruses inversely correlate with the genomic size.

Undifferentiated keratinocytes control growth, morphology, and antigen expression of normal melanocytes through cell-cell contact.

BACKGROUND: Melanocytes in the normal human epidermis are generally dendritic and neither proliferate nor express melanoma-associated antigens. In culture, on the other hand, melanocytes are bi- to tripolar, proliferate with 2 to 4 day doubling times, and express melanoma-associated antigens. This observation prompted us to investigate the regulatory role of keratinocytes for growth, morphology, and antigen expression of melanocytes. EXPERIMENTAL DESIGN: Melanocytes and keratinocytes were cultured under three different co-culture conditions: (a) separated by a semiporous membrane, (b) in monolayer cultures allowing direct contact between cells, and (c) in three-dimensional epidermal reconstructs. RESULTS: Melanocytes separated from keratinocytes by semiporous membranes remained di- and tripolar and could not proliferate in medium optimal for keratinocytes. When cell-cell contact was established between melanocytes and undifferentiated, but not differentiated, keratinocytes, melanocytes proliferated at a rate similar to keratinocytes and they developed multiple dendrites. In co-cultures allowing the multi-layered growth of keratinocytes, melanocytes were nonproliferative when juxtaposed to undifferentiated keratinocytes in the basal layer, but proliferated when surrounded by differentiated keratinocytes in the intermediate and upper layers. Expression of melanoma-associated antigens on melanocytes decreased to similar levels as in normal skin when melanocytes were in direct contact with undifferentiated, but not differentiated, keratinocytes. CONCLUSIONS: Undifferentiated, but not differentiated, keratinocytes control growth, morphology, and antigen expression of melanocytes through direct cell-cell contact. These results suggest that the phenotypic characteristics of nevus and melanoma cells in the dermis, i.e., proliferation and expression of tumor-associated antigens, may be due to their loss of contact with undifferentiation keratinocytes.

Growth and invasion of human melanomas in human skin grafted to immunodeficient mice.

An orthotopic model of human melanoma was developed in which malignant cells were injected into human skin grafted to nude and SCID mice. Melanoma cells proliferated and invaded the human skin grafts with characteristic patterns. Three of six melanomas grew as multiple nodules and infiltered the grafts without major architectural changes in the dermis, whereas the others invaded the dermis along collagen fibers with prominent endothelial vessels. By contrast, melanoma cells inoculated into mouse skin grew as diffusely expanding nodules that did not invade the murine dermis. In human skin grafts, human melanoma cells were angiogenic for human blood vessels, and murine vessels were only found at the periphery of grafts. Tumor cells invaded the human vessels, and four out of seven cell lines metastasized to lungs, suggesting that this model is useful to determine in vivo the interactions between normal and malignant human cells.

Spontaneous and induced differentiation of human melanoma cells.

Malignant melanoma cells can differentiate spontaneously in vivo and in vitro into cells with a finite lifespan. Analysis of differentiating cells from primary melanomas in culture revealed a flat, fibroblast-like morphology and expression of the fibroblast-associated marker leucine aminopeptidase (LAP). Differentiation was also observed in a minor sub-population of permanent cell lines derived from metastatic lesions. An experimental model of melanoma cell differentiation was then developed, using the pyrimidine analog bromodeoxyuridine (BUdR). BUdR-treated cells had a flat morphology, were contact-inhibited, had up to 20-fold increased surface area, expressed LAP, no longer proliferated anchorage-independently in soft agar, and 3 out of 4 cell lines were non-tumorigenic in athymic nude mice. Our results show that models of differentiation of melanoma cells can be established that help to define pathways of differentiation.

Growth regulation of cultured human nevus cells.

Cells isolated from congenital melanocytic nevi and cultured in vitro have growth characteristics that resemble their premalignant stage in situ. A serum-free, chemically defined medium has been developed that allows continuous growth of established nevus cultures for up to several months. Like primary melanoma cells, nevus cells in high-calcium-containing W489 medium require insulin for growth. In contrast to melanoma cells, nevus cells in serum-free medium require the presence of alpha-melanocyte-stimulating hormone, which enhanced intracellular levels of cyclic adenosine monophosphate. In contrast to the requirements of normal human melanocytes from newborn foreskin, congenital nevus cells grow with less dependency on basic fibroblast growth factor (bFGF). Nevus cultures contain bFGF-like activity, and they express bFGF mRNA. Nevic cells of compound nevi also express bFGF mRNA in situ but only in the junctional areas. These results indicate that bFGF plays an important growth regulatory role for nevus cells in vitro and in vivo.

Platelet-derived growth factor (PDGF) in oncogenesis: development of a vascular connective tissue stroma in xenotransplanted human melanoma producing PDGF-BB.

Human WM9 melanoma cells, previously shown to be devoid of PDGF expression, were stably transfected with a PDGF-B cDNA under the transcriptional control of a cytomegalovirus promoter. Northern blot analysis revealed high expression of an mRNA of the expected size in the PDGF-B-transfected cells. Synthesis and secretion of PDGF-BB was confirmed by immunoprecipitation. Furthermore, conditioned medium from PDGF-B-transfected cells contained a mitogenic activity for fibroblasts. For analysis of tumor growth in vivo, cells of each type were injected subcutaneously into BALB/c nu/nu mice. Tumors from mice injected with WM9 cells transfected with the vector only contained large necrotic areas; only scant blood vessels with narrow lumina were observed. No connective tissue was present. In the tumors from PDGF-B-transfected WM9 cells, nests of tumor were divided by connective tissue septa. An abundance of blood vessels was observed in the connective tissue septa and within the tumor cell nests. There was a complete absence of necrosis in these tumors. The present results suggest that tumor-derived PDGF-BB is a potent mediator of connective tissue stroma formation. The connective tissue framework that is generated in response to PDGF-BB may form a solid support for newly formed blood vessels and, thereby, facilitate the formation of a functional vascular system in the tumor.

Cytokine-induced expression of transforming growth factor-alpha and the epidermal growth factor receptor in neonatal skin explants.

Hyperproliferative diseases of the epidermal keratinocytes, such as psoriasis vulgaris, are characterized by overexpression and altered distribution of the epidermal growth factor/transforming growth factor (EGF/TGF)-alpha receptor, and of TGF-alpha itself. It is believed that overexpression of this lignad/receptor system contributes to the hyperproliferative state of keratinocytes in an autocrine fashion. However, little is known about the factors that regulate expression of the EGF/TGF-alpha receptor, as well as expression of TGF-alpha in stratified epithelium. We examined modulation of the immunoreactive EGF/TGF-alpha receptor and TGF-alpha expression in normal neonatal foreskin explants by a variety of cytokines present in psoriatic lesions. Human (hu) recombinant (r) tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma induced EGF/TGF-alpha receptor and TGF-alpha expression by keratinocytes as determined by immunohistochemistry. Neutralizing antibodies to TNF-alpha and IFN-gamma inhibited upregulation of EGF/TGF-alpha receptors and TGF-alpha by the respective cytokines. Interleukin (IL)-8 induced expression of TGF-alpha, but not of its receptor. Other cytokines (TNF-beta, IFN-beta, IL-1 alpha, IL-2, IL-3, IL-5, IL-6, granulocyte/macrophage colony-stimulating factor, and macrophage colony-stimulating factor) did not alter the expression patterns of EGF/TGF-alpha receptors or TGF-alpha in normal neonatal skin explants. These experiments demonstrate that specific cytokines known to be present in psoriatic lesions can induce normal epidermis to express TGF-alpha and its receptor in a pattern similar to that observed in psoriatic skin.

Phenotypes and interactions of human melanocytes and keratinocytes in an epidermal reconstruction model.

The morphologic and antigenic phenotype of normal human melanocytes and keratinocytes was investigated in monolayer and 3-dimensional cultures in an effort to develop an epidermal model that resembles the normal human epidermis. When cultured for several passages in optimal growth medium, pure cultures of either cell type could be established as demonstrated by light and electron microscopy and with monoclonal antibodies defining melanocyte- and keratinocyte-associated antigens. Three-dimensional growth of keratinocytes on polycarbonate filters was induced by increasing calcium concentrations in the culture medium and exposing cultures to air. After 30 to 35 days incubation, the 3-dimensional keratinocyte cultures reached a total of 12 to 25 layers and keratinocytes of various stages of differentiation formed three morphologically and antigenically different strata. The basal layer of these constructs consisted of ovoid cells with desmosomes and hemidesmosome-like structures. These cells expressed low molecular weight cytokeratins similar to basal cells in situ. The intermediate layer, representing the stratum spinosum in situ, contained flat cells with keratohyaline granules and many desmosomes. These cells expressed gp 80 kilodaltons, gp 40 to 50 kilodaltons, involucrin, and filaggrin. The upper layer, the stratum corneum equivalent, contained large, flattened cells with keratohyaline granules. The majority of these cells were anucleate. When melanocytes were cocultured with keratinocytes in monolayer or in epidermal reconstructs, they assumed a multidendritic morphology and donated pigment to surrounding keratinocytes. The majority of pigmented cells localized singly within the basal layer of the reconstructs and their dendrites were intimately associated with keratinocyte plasma membranes. Pigment donation to keratinocytes appeared to occur through the uptake of melanosome-containing dendrite fragments and phagocytosis of individual melanosomes by keratinocytes. It is hypothesized that keratinocytes produce unique microenvironmental factors that regulate the melanocytic phenotype.

Growth-regulatory factors for normal, premalignant, and malignant human cells in vitro.

Normal human cells, cells from nonmalignant proliferative lesions, and primary and metastatic tumor cells can be maintained in vitro and analyzed for requirements for growth in chemically defined media. The human melanocytic cell system with normal melanocytes, precursor nevus cells, and primary and metastatic melanoma cells has been extensively studied for the phenotypic properties of the cells, including their requirements for exogenous growth factors and other mitogens. In high calcium-containing W489 medium, normal melanocytes require four supplements: IGF-I (or insulin); bFGF, TPA, and alpha-MSH. Nevus cells are largely independent of bFGF. Depletion of TPA from medium is not as detrimental to nevus cells as it is to melanocytes, but the phorbol ester is still essential for maintenance of the typical nevic phenotype. Primary melanoma cells require at least one growth factor, IGF-I (or insulin), for continuous proliferation. On the other hand, metastatic cells of melanoma as well as of carcinomas of colon and rectum, bladder, ovary, and cervix are able to proliferate after a short adaptation period in medium depleted of any growth factors and other proteins. Doubling times of metastatic tumor cells in protein-free medium are only 30-60% longer than in FCS-containing medium. The growth autonomy of human tumor cells is apparently due to the endogenous production of growth factors. Likely candidates for autocrine growth stimulation of human tumor cells are TGF-alpha, TGF-beta, and PDGF. Melanoma and colorectal carcinoma cells express functional EGF/TGF-alpha receptors, and produce TGF-alpha, indicating that this growth factor is produced for autocrine stimulation. In addition to the use of anti-growth factor antibodies, other strategies for the inhibition of autocrine growth stimulation include mAbs to growth factor receptors, soluble receptors, receptor-mimicking antiidiotype antibodies, and active immunization against growth factors. Whether any of these therapeutic approaches is clinically feasible will need to be determined in extensive preclinical investigations.