Detection of cytokine gene expression in human monocytes and lymphocytes by fluorescent in situ hybridization in cell suspension and flow cytometry

OBJECTIVES: Previous reports indicate that up to 10% of patients with localized renal cell carcinoma have direct intracaval neoplastic extension. Many patients with locally confined tumors and small intracaval tumor extensions can be surgically cured. Few studies have documented long-term survival after radical surgery for renal cell carcinoma involving higher vena caval tumor extension. We report the follow-up of 34 consecutive patients undergoing radical nephrectomy and intrahepatic or supradiaphragmatic intracaval thrombectomy for renal cell carcinoma. METHODS: From October 1982 through January 1993, 34 consecutive patients with a mean age of 60 years were identified as having clinical Stage T3 renal cell carcinoma (mean diameter 9.5+/-4.0 cm) with intrahepatic (41%) or supradiaphragmatic (59%) intracaval neoplastic extension. Patients underwent radical nephrectomy with intrahepatic caval thrombectomy (38%) or supradiaphragmatic caval thrombectomy using cardiac bypass with hypothermia and circulatory arrest (62%). Clinical outcome was assessed during a mean follow-up of 30 months (range 1 to 182). RESULTS: A total of 24 (71%) of 34 tumors demonstrated capsular penetration, and 22 (65%) of 34 had significant perinephric extension into Gerota's fascia by pathologic analysis. Metastatic disease was identified in 35% of patients either at the time of surgery or by pathologic analysis. Using Kaplan-Meier actuarial analysis, the likelihood of survival for all 34 consecutive patients after surgery was 68% (95% confidence interval [CI] 49% to 81%) at 1 year, 32% (95% CI 18% to 48%) at 2 years, 14% (95% CI 5% to 28%) at 5 years, and 9% (95% CI 2% to 24%) at 10 years. Neither capsular penetration, perinephric extension, the level of intracaval extension of tumor, nor the use of cardiopulmonary bypass significantly affected survival. CONCLUSIONS: In patients with renal cell carcinoma and intrahepatic or supradiaphragmatic intracaval extension of tumor, the presence of metastases is a frequent occurrence and, if present, greatly diminishes survival. Improvements in the preoperative detection of occult metastases are needed if surgery alone is to improve survival.     

Induction of interleukin-10 by human immunodeficiency virus type 1 and its gp120 protein in human monocytes/macrophages

In this study, we evaluated the effects of human immunodeficiency virus type 1 (HIV-1) and its gp120 protein on interleukin-10 (IL-10) expression in cultured human monocytes/macrophages. Infection of either 1-day monocytes or 7-day monocyte-derived macrophages with HIV-1 strain Ba-L resulted in clear-cut accumulation of IL-10 mRNA at 4 and 24 h. Likewise, treatment of these cells with recombinant gp120 induced IL-10 mRNA expression and caused a marked increase in IL-10 secretion. Monoclonal antibodies to gp120 strongly inhibited recombinant gp120-induced IL-10 secretion by monocytes/macrophages. Moreover, the addition of IL-10 to monocytes/macrophages resulted in a significant inhibition of HIV-1 replication 7 and 14 days after infection. On the whole, these results indicate that HIV-1 (possibly through its gp120 protein) up-regulates IL-10 expression in monocytes/macrophages. We suggest that in vivo production of IL-10 by HIV-primed monocytes/macrophages can play an important role in the early response to HIV-1 infection.     

Consequences of gamma-irradiation on inflammatory cytokine regulation in human monocytes/macrophages

We have investigated mitochondrial DNA (mtDNA) mutagenesis in the laboratory mouse. Using a nested PCR method for quantification, the absolute frequency, tissue distribution and rate of increase of mitochondrial deletion mutations was determined. Multiple deletions arise in brain, cardiac muscle and kidney tissues: deletions occur most frequently at regions of directly repeated mtDNA homology. Deletion frequencies rose by 2.5 x 10(5), 6300- and 4000-fold in heart, brain and kidney, respectively, between young and old mice. The rates of mtDNA mutation accumulation in mouse and human hearts are modeled well by exponential equations, with r-values of 0.96 and 0.97, and mutations rose much faster in mouse than human mtDNA per unit time. Thus, maintenance of the human mitochondrial genome is much better than that of mice, consistent with the higher rate and final extent of total DNA repair in humans than mice, that has been observed by others and consistent with the predictions of the disposable soma model of aging. A comparison of mtDNA mutagenesis from cardiocytes vs. whole heart tissue was undertaken. Deletion mutations were observed to be 100-fold lower in DNA prepared from isolated cardiocytes than from whole heart homogenates, consistent with a model of uneven mtDNA mutation accumulation.     

Mediation of entry of human immunodeficiency virus-1 into alveolar macrophages by CD4 without facilitation by surfactant-associated protein-A

The mechanism of uptake of human immunodeficiency virus-1 (HIV-1) into alveolar macrophages (AM), freshly isolated blood monocytes (MN), and cultured MN (CM) was investigated focusing on the role of CD4 and of surfactant-associated protein A (SP-A). By radioimmunoassay which obviated the problems of auto- and nonspecific fluorescence of more differentiated macrophages, each of the macrophage populations studied expressed CD4. Semiquantitative polymerase chain reaction was performed to assess uptake of HIV-1(JR-FL) into cells. OKT4a (directed against CD4) blocked uptake of HIV-1 into CM, AM, and MN by 67 to 100%. OKT4 (directed against another epitope of CD4) had a smaller and less consistent effect (0-90%), and control antibodies showed minimal effects and only at supersaturating concentrations. SP-A had no effect on uptake of HIV-1 into AM. SP-A also had no consistent effect on production of HIV-1(JR-FL) by AM infected in vitro (p24 antigen ELISA). Thus CD4 is the major receptor for HIV-1 in mononuclear phagocytes, including AM, and SP-A does not modulate entry.     

Inhibition of human immunodeficiency virus-1 protease by a C2-symmetric phosphinate. Synthesis and crystallographic analysis

The human immunodeficiency virus type 1 (HIV-1) protease is a potential target of acquired immune deficiency syndrome (AIDS) therapy. A highly potent, perfectly symmetrical phosphinate inhibitor of this enzyme, SB204144, has been synthesized. It is a competitive inhibitor of HIV-1 protease, with an apparent inhibition constant of 2.8 nM at pH 6.0. The three-dimensional structure of SB204144 bound to the enzyme has been determined at 2.3-A resolution by X-ray diffraction techniques and refined to a crystallographic discrepancy factor, R (= sigma parallel F(o) magnitude to - Fc parallel/sigma magnitude of F(o)), of 0.178. The inhibitor is held in the enzyme active site by a set of hydrophobic and hydrophilic interactions, including an interaction between Arg8 and the center of the terminal benzene rings of the inhibitor. The phosphinate establishes a novel interaction with the two catalytic aspartates; each oxygen of the central phosphinic acid moiety interacts with a single oxygen of one aspartic acid, establishing a very short (2.2-2.4 A) oxygen-oxygen contact. As with the structures of penicillopepsin bound to phosphinate and phosphonate inhibitors [Fraser, M. E., Strynadka, N. C., Bartlett, P. A., Hanson, J. E., & James, M. N. (1992) Biochemistry 31, 5201-14], we interpret this short distance and the stereochemical environment of each pair of oxygens in terms of a hydrogen bond that has a symmetric single-well potential energy curve with the proton located midway between the two atoms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumor necrosis factor-alpha mRNA and protein in endometrial tumors: analysis by in situ hybridization and immunocytochemistry

Abnormal expression of polypeptide growth factors and their receptors is closely associated with tumorigenic transformation. In this study tumor necrosis factor-alpha (TNF-alpha) mRNA and protein were analyzed in polyps and proliferative lesions of endometrium as well as in low and high grade endometrial tumors by using in situ hybridization and immunocytochemistry. All samples contained products of the TNF-alpha gene. Histochemical scores (HS), which reflect the proportion of cells positive for TNF-alpha message or protein and the intensities of the signals, were higher for epithelial than for stromal cells. Benign lesions (endometrial polyps) contained little TNF-alpha mRNA or protein, whereas specific message was abundant in proliferative lesions (hyperplasia, adenofibroma). Although neoplastic cells in both low and high grade endometrial tumors contained TNF-alpha mRNA, two major differences were observed: HS for TNF-alpha mRNA were significantly less in low grade than in high grade neoplasms, and TNF-alpha message was restricted to the nucleus in low grade adenocarcinoma cells but was abundant in the cytoplasm of high grade tumor cells. In contrast to cells in benign and proliferative lesions, TNF-alpha protein scores in endometrial tumor cells were inversely rather than positively correlated with TNF-alpha mRNA scores. Collectively, the findings in this study are consistent with the postulate that TNF-alpha is useful to endometrial tumor cells and suggest that production may increase as cells diverge from normal.     

Temporary seronegativity in a human immunodeficiency virus type 1-infected man

In addition to financial constraints there are significant social, educational, moral and religious barriers to the prevention and treatment of reproductive tract infections in rural India. A pilot project aimed at achieving progress in this field is reported below.     

Cytogenetic analysis of BC2, a new human hepatoma cell line, by fluorescent in situ hybridization

Phosphodiesterases (PDEs) are key enzymes involved in the regulation of intracellular cyclic nucleotide metabolism. The aim of the present study was to identify and to characterize the PDE isoenzymes present in the human detrusor smooth muscle. Human detrusor PDE isoenzymes were separated by Q-Sepharose anion exchange and calmodulin-agarose affinity chromatography and characterized upon their kinetic characteristics and their sensitivity to allosteric modulators and inhibitors. All five presently known PDE isoenzyme families were identified: one high-affinity, low-Km calcium/calmodulin-stimulated PDE I with a slight preference for cGMP over cAMP, one cGMP-stimulated PDE II, one cGMP-inhibited PDE III, one cAMP-specific PDE IV and one cGMP-specific PDE IV. All five known PDE isoenzyme families exist in human detrusor smooth musculature. The kinetic characteristics, together with functional in vitro studies, suggest that the PDE I may be of importance in the intracellular regulation of the human detrusor smooth muscle tone.     

Electronmicroscopic study of human herpesvirus 6-infected human T cell lines superinfected with human immunodeficiency virus type 1

Melatonin release by chick cultured pineal cells increases during the dark periods and decreases during the light periods under light-dark cycles, and this rhythmic secretion is maintained under constant conditions with a period of almost 24 hr. The mechanisms by which the circadian oscillator drives the melatonin rhythm under constant conditions have not been elucidated enough. We examined the possibility that cyclic AMP-dependent protein kinase A is involved in the subjective nocturnal increase in melatonin release by chick pineal cells cultured under constant darkness. The subjective nocturnal increase of melatonin release was suppressed dose dependently by H8 (protein kinase inhibitor) and H89 (specific protein kinase A inhibitor), but not by calphostin C (specific protein kinase C inhibitor) in static cell cultures. In a cell perfusion experiment, 9 hr pulses of H8 and H89 starting at ZT 9 (CT 11.2) hr suppressed the subjective nocturnal increase in melatonin rhythm in dose-dependent manner without causing a phase shift. An intracellular Ca2+ chelator, O,O'-bis(2-aminophenoxy)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), and extracellular Ca2+ chelators, O,O'-bis(2-aminophenoxy)ethyleneglycol-N,N,N',N'-tetraacetic acid tetrapotassium salt hydrate (BAPTA) and ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), suppressed both the subjective nocturnal increases in melatonin release and cAMP levels dose dependently. This direct evidence strongly supports the hypothesis that cAMP-dependent protein kinase A may be involved in the subjective nocturnal increase in melatonin release by chick pineal cells and that intracellular Ca2+ plays an important role in pineal adenylate cyclase activation.     

Cytokine repertoire during maturation of monocytes to macrophages within spheroids of malignant and non-malignant urothelial cell lines

Terminal maturation of human blood monocytes to macrophages (MAC) in vivo is believed to be important for the morphology, antigen expression and functional activity of the resulting MAC population. This process is modulated by the specific tissue micro-environment to which blood monocytes migrate upon leaving the vasculature. Tumor-associated macrophages (TAM) are a special type of MAC, and little is known about the modulating capacity of the tumor environment on monocyte-to-MAC differentiation. By co-culturing 3-dimensional multicellular spheroids (MCS) of the urothelial-bladder-carcinoma cell lines J82 and RT4 with human monocytes/MAC we generated TAM in vitro. For comparison, monocytes/MAC were co-cultured with the non-tumorigenic urothelial cell line HCV29. The effects on monocyte differentiation were analyzed, particularly with respect to cytokine release. Monocyte maturation was modulated within the tumor spheroid dependent upon the tumor cell type. Monocytes co-cultured with MCS of the poorly differentiated J82 carcinoma spontaneously produced high amounts of IL-1beta and IL-6, but only low amounts of TNF-alpha, which could be further increased by the addition of LPS. This cytokine pattern is characteristic for monocytes and remained constant for up to 8 days in J82-MCS co-cultures. However, in RT4-MCS and HCV29-MCS co-cultures, the initial cytokine pattern changed and after 8 days corresponded well to that of MAC differentiated in vitro without tumor contact. In addition to functional parameters, we analyzed the morphology of J82-MCS-TAM and found that they displayed a monocyte-like morphology. Our data indicate that (1) tumor cells can influence monocyte-to-MAC differentiation, giving rise to TAM with monocyte-specific phenotypic properties; and (2) this capacity is dependent on the type of tumor cell.     

Combined genotypes of CCR5, CCR2, SDF1, and HLA genes can predict the long-term nonprogressor status in human immunodeficiency virus-1-infected individuals

Human immunodeficiency virus (HIV)-1-infected long-term nonprogressors (LT-NP) represent less than 5% of HIV-1-infected patients. In this work, we tried to understand whether combined genotypes of CCR5-triangle up32, CCR2-64I, SDF1-3'A and HLA alleles can predict the LT-NP status. Among the chemokine receptor genotypes, only the frequency of the CCR5-triangle up32 allele was significantly higher in LT-NP compared with the group of standard progressors. The predominant HLA alleles in LT-NP were HLA-A3, HLA-B14, HLA-B17, HLA-B27, HLA-DR6, and HLA-DR7. A combination of both HLA and chemokine receptor genotypes integrated in a multivariate logistic regression model showed that if a subject is heterozygous for CCR5-triangle up32 and homozygous for SDF1 wild type, his odds of being LT-NP are increased by 16-fold, by 47-fold when a HLA-B27 allele is present with HLA-DR6 absent, and by 47-fold also if at least three of the following alleles are present: HLA-A3, HLA-B14, HLA-B17, HLA-DR7. This model allowed a correct classification of 70% of LT-NPs and 81% of progressors, suggesting that the host's genetic background plays an important role in the evolution of HIV-1. The chemokine receptor and chemokine genes along with the HLA genotype can serve as predictors of HIV-1 outcome for classification of HIV-1-infected subjects as LT-NPs or progressors.     

Sugar transport and glut transporter expression in a variety of human immunodeficiency virus-1 (HIV-1) chronically infected target cell lines

In this study, sugar transport and the cellular content of the human Glut 1 and 3 glucose transporters were ascertained in uninfected and chronically HIV-infected Jurkat and H9 cell lines (T-cell lines) and U937 cells (a promonocytic cell line). Sugar transport was determined by monitoring 2-deoxy glucose uptake (2DG) and glut transporter content was determined by Western analysis. Although 'acute' HIV infection of H9 cells led to increased cellular transport activity and Glut 3 transporter content, chronic HIV infection exhibited no significant differences in sugar transport in any of the cell types investigated whether log or stationary phase cultures were employed. When uninfected and chronically HIV-infected cell lines were compared, all cell lines expressed the Glut 1 transporter, however, significant differences in Glut 1 transporter content were not observed. The Glut 3 transporter which could only be detected in the H9 cell line exhibited no differences in Glut 3 content in uninfected or chronically HIV-infected cells (2.1 +/- 0.6 versus 3.8 +/- 2.1 x 10(-3) arbitrary units/microgram protein). A trend towards lower amino acid uptake was seen in the chronically HIV-infected cells but this was not significantly different from uninfected cell cultures. The data indicate that: (1) glucose transport and the Glut 1 and 3 transporters are not increased in cells chronically infected with HIV-1 and (2) the expression of the Glut 3 sugar transporters is not the same in all target cells.     

Assignment of the dynamin-1 gene (DNM1) to human chromosome 9q34 by fluorescence in situ hybridization and somatic cell hybrid analysis

The dynamins are recently discovered GTP-binding proteins postulated to mediate the scission of clathrin-coated vesicles at the plasma membrane. Of the three known mammalian dynamins, dynamin-1 (DNM1) appears to be particularly important for the formation of synaptic vesicles at presynaptic nerve termini. To investigate the possibility that mutations in the DNM1 gene cause a human disease, we determined the chromosomal localization of human DNM1. We conclude from fluorescence in situ hybridization and from the analysis of somatic cell hybrids that the map position in 9q34. This region has syntenic homology with mouse chromosome 2p, in agreement with the map position of the mouse DNM1 gene [see accompanying article by Klocke et al. (1997, Genomics 41:290-292)]. We discuss the potential relevance of the human DNM1 localization to diseases that were mapped genetically to the same chromosomal region.     

Activation of the human immunodeficiency virus-1 long terminal repeat by respiratory burst oxidants of neutrophils

Unipolar localization of IcsA on the surface of Shigella flexneri is required for efficient formation of actin tails and protrusions in infected eucaryotic cells. Lipopolysaccharide (LPS) mutations have been demonstrated to affect either the establishment or the maintenance of IcsA in a unipolar location, although the mechanism is unknown. In order to analyze the contribution of virulence plasmid determinants on the unipolar localization of IcsA, we examined the localization of IcsA expressed from a cloned plasmid copy in two different genetic backgrounds. The localization of IcsA was first examined in a virulence plasmid-cured derivative of the wild-type S. flexneri 2a isolate 2457T. This approach examined the contribution of virulence plasmid-borne factors, including the previously identified virulence plasmid-borne protease that is responsible for cleaving IcsA in the outer membrane and releasing the 95-kDa secreted form from the cell surface. IcsA localization in a related but nonpathogenic Escherichia coli strain expressing LPS of the O8 serotype was also examined. IcsA surface presentation in both of these genetic backgrounds continued to be unipolar, demonstrating that virulence plasmid-borne determinants are not responsible for unipolar localization of IcsA. The unipolar localization of IcsA in the E. coli background suggests that a common pathway that allows IcsA to be spatially restricted to one pole on the bacterial cell surface exists in Shigella and E. coli.     

Expression of cell adhesion molecules at the surface of in vitro human immunodeficiency virus type 1-infected human monocytes: relationships with tumor necrosis factor alpha, interleukin 1beta, and interleukin 6 syntheses

Further evidence suggests that cell adhesion molecules (CAMs) expressed on the surface of human immunodeficiency virus type 1 (HIV-1)-infected cells are regulated during lentiviral infection. To address this hypothesis we have investigated the kinetic pattern of CAM expression at the surface of HIV-1Ba.L-infected human monocytes during the first 72 hr of infection. A significantly lower expression of CD18 and CD54 as well as a decrease in CD44 expression level were observed at the surface of infected monocytes when compared with mock-infected cultures. No modification of CD11a, CD11b, CD11c, CD58, and CD62L expression was detected. Except for CD18, the expression of which at the cell surface is decreased, no modification of CD44 and CD54 expression was observed after heat-inactivated HIV-1 treatment of monocytes. Investigation of soluble forms of CAMs (sCAMs) and cytokine production in the culture supernatants of infected monocytes showed a peak of sCD44, TNF-alpha, IL-1beta, and IL-6 release between 2 and 24 hr after infection. Treatment of monocytes with monoclonal antibodies (MAbs) against CAMs showed that engagement of some CAMs may trigger TNF-alpha and IL-1beta production. In addition, pretreatment of infected monocytes with a TNF-alpha synthesis inhibitor, RP 55778, or with MAbs directed against IL-1beta, confirmed the role of TNF-alpha and IL-1beta in the regulation of CD18, CD44, and CD54 expression.     

Interaction of human cytomegalovirus with monocytes/macrophages: a love-hate relationship

This review addresses the complex interactions of human cytomegalovirus (CMV) with the phagocytic mononuclear cell system. Certain aspects may be supplemented with observations made in animal systems. An attempt will be made to summarize information acquired through in vivo studies about the distribution of CMV in the mononuclear phagocyte cells, the state of the virus in such cells and the reactivation of virus in this setting. Much information has been gained through in vitro experimentation attempting to elucidate the infectability of and replication in monocytes/macrophages, and the role played by the cellular differentiation in these events. The influence of CMV infection on the generation of macrophages and granulocytes from bone marrow progenitors will also be examined. The modifications of host cell function induced by CMV infection, notably the production of cytokines and antigen presentation will be examined. The possible significance of CMV infection of monocytes/macrophages relative to viral dissemination, immunosuppression, and hematological modifications associated with CMV infection will be discussed.