Production and expression of RANTES (CCL5) by human disc cells and modulation by IL-1-β and TNF-α in 3D culture

Chemokines act as important secondary inflammatory mediators which are released by cells in response to a variety of stimuli. Chemokines bind to cell surface receptors and act as second-order cytokines with specialized functions in inflammation. The role of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted) (also called CCL5 (chemokine (C–C motif) ligand 5)) has received little attention to date in disc tissue. Microarray analyses of lumbar disc annulus tissue revealed that RANTES expression was significantly upregulated in more degenerated Thompson grades IV and V discs compared to expression levels in grades I, II and III discs (p = 0.032). Immunolocalization confirmed the presence of RANTES in the annulus and nucleus of the disc, and localized the RANTES receptors CCR1, CCR3 and CCR5 to cells in the disc. In vitro studies with IL-1-β and TNF-α challenges, both proinflammatory cytokines resulted in elevated levels of RANTES in conditioned media (p < 0.01); TNF-α exposure, however, produced significantly greater levels than did IL-1alpha (p < 0.0001), suggesting a differential regulation by TNF-α. Local production of RANTES in vivo by annulus and nucleus cells, and in vitro induction of RANTES by proinflammatory cytokines suggest that disc cells are primary effector cells as well as target cells, and thus can mediate physiological immune-related processes during disc degeneration by both autocrine and paracrine signaling.     

Isolation of integrin αIIbβ3 (glycoprotein IIb-IIIa) from human platelets

Summary This report describes a method for the purification of the platelet fibrinogen receptor, integrin _IIb3. The procedure involves lectin affinity chromatography on concanavalin-A agarose followed by heparin-agarose and gelatin-agarose. The integrin is further purified by gel filtration chromatography on Sephacryl S-300. Substantial amounts of purified _IIb3 are obtained (5 mg/10 units of platelets) and the procedure is easily scaled up or down.     

Expression of ADAMs (“a disintegrin and metalloprotease”) in the human lung

In view of the associations of “a disintegrin and metalloprotease” (ADAM) with respiratory diseases, we assessed the expression of various ADAMs in human lung tissue. Lung tissue was obtained from nine individuals who underwent surgery for lung cancer or underwent lung transplantation for emphysema. Also, 16HBE 14o- (human bronchial epithelial) and A549 (alveolar type II epithelium-like) cell lines were used. Immunohistochemistry was performed with antibodies recognizing different ADAM domains. The ADAMs were typically distributed over the bronchial epithelium. ADAM8 and ADAM10 were expressed diffusely in all layers of the epithelium. ADAM9, ADAM17, and ADAM19 were predominantly expressed in the apical part of the epithelium, and ADAM33 was predominantly and strongly expressed in basal epithelial cells. In smooth muscle, ADAM19 and ADAM17 were strongly expressed, as was ADAM33, though this expression was weaker. ADAM33 was strongly expressed in vascular endothelium. All ADAMs were generally expressed in inflammatory cells. The typical distribution of ADAMs in the lung, especially in the epithelium, is interesting and suggests a localized function. As most ADAMs are involved in release of (pro-) inflammatory mediators and growth factors, they may play an important role in the first line of defense and in initiation of repair events in the airways.     

Characterization and V(H) sequences of human monoclonal anti-F(ab')(2) autoantibodies from normals and Sjögren's syndrome patients

To investigate the genetic background of anti-F(ab')(2) autoantibodies and the mechanism behind their production we have analyzed 10 human monoclonal antibodies directed against IgG F(ab')(2) and IgG Fab. They were all derived from peripheral blood by the EBV/hybridoma technique. Eight were from three healthy individuals and two from two patients with primary Sj?gren's syndrome (pSS). They react with epitopes on distinct regions of IgG, including epitopes present on or near the hinge of IgG, epitopes on the Fd gamma, and an antigenic determinant(s) present on lambda light chains. These determinants are either exposed on the intact IgG molecule or revealed following pepsin or papain digestion. The V(H) germline gene repertoire used is diverse and with considerable overlap with that used by rheumatoid factors (RF). The two IgG antibodies from normals are extensively mutated (13 and 24 mutations/V(H)), but with a replacement to silent mutation ratio in the CDR(H)1 + 2 of only 3.7. The IgM antibodies from normals are also heavily mutated (mean 10 mutations/V(H)). This suggests that anti-F(ab')(2) from normals are generated by an antigen-driven somatic hypermutation mechanism. In contrast, the two IgM antibodies from pSS are virtually unmutated in both V(H) and V(L). Together with published data of pSS RF and anti-Ro 52-kDa sequences (1-3), this suggests that there is an expanded population of na?ve B cells with autoantibody specificities in the peripheral blood of pSS patients.     

Deletion of the D domain of the human parainfluenza virus type 3 (HPIV3) PD protein results in decreased viral RNA synthesis and beta interferon (IFN-β) expression

Parvoviridae is a family of small non-enveloped viruses and divided into two subfamilies. The family members infect a wide range of organisms from insects to humans and some of the members (e.g., nonpathogenic adeno-associated viruses) are effective gene therapy delivery vectors. We detailed the synonymous codon usage pattern of Parvoviridae family from the available 58 sequenced genomes through multivariate statistical methods. Our results revealed that nine viruses showed some degree of strong codon bias, and the others possessed a general weak trend of codon bias. ENc-plot and neutrality plot results showed that selective pressure dominated over mutation in shapes coding sequence’s composition. The overall GC content and GC content at the third synonymous codon position were the principal determinants behind the variations within the codon usage patterns, as they both significantly correlated with the first axis of correspondence analysis. In addition, gene length had no direct influence on the codon usage pattern. Densovirinae subfamily and Parvovirinae subfamily possessed nine identical preferred codons, though most of the two subfamilies codon usage frequencies were significantly different. The result of cluster analysis based on synonymous codon usage was discordant with that of taxonomic classification. Adeno-associated viruses formed a separated clade far from other Parvoviridae members in the dendrogram. Thus, we concluded that natural selection rather than mutation pressure accounts for the main factor that affects the codon bias in Parvoviridae family.     

Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces human CC- and CXC-chemokines in vitro and in vivo

Both CC- and CXC-chemokines are known to be potent leucocyte activators and chemoattractants and play important roles in inflammatory responses. However, chemokine response to bacillus Calmette-Guérin (BCG) infection remains incompletely defined. In this study, we investigated human CC- [macrophage-derived chemokine (MDC), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha and eosinophil chemoattractant activity (eotaxin)] and CXC-interferon-inducible protein (IP)-10 chemokine production in response to BCG stimulation. BCG efficiently induced all chemokines tested in the urine of four bladder cancer patients undergoing intravesical BCG immunotherapy. The peak urinary chemokine responses occurred generally between the fourth and sixth weekly treatment, except eotaxin, which was less predictable. To evaluate the effect of BCG on induction of chemokines in vitro, urothelial cell lines and peripheral blood mononuclear cells (PBMCs) were used. Although BCG induced no or marginal chemokines from urothelial SV-HUC-1, RT4 and T24 cells, BCG-derived cytokines [interleukin (IL)-1beta, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha] induced all chemokines tested except eotaxin from these cell lines. BCG also efficiently induced all chemokines tested except eotaxin from PBMCs of both BCG-naive and BCG-vaccinated subjects. MCP-1 and MIP-1alpha emerged at 4-5 h post-BCG exposure (early chemokines); IP-10 elevated at day 1 and peaked at day 2 (intermediate chemokine); and MDC elevated at day 1 and peaked at day 7 (late chemokine). This kinetic pattern was paralleled with that of BCG-induced cytokines [early: TNF-alpha; intermediate: IL-6 and IL-10; and late: IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF)]. Taken together, these results indicate that BCG directly or indirectly induces human CC- and CXC-chemokine production, which may represent one of the mechanisms by which BCG exerts its anti-tumour activity.     

Effects of smoking on benzo(α)pyrene- and glutathione-metabolizing enzymes in human lung tissue

Summary The influence of smoking on the metabolism of benzo()pyrene and glutathione was investigated in 190 patients with primary bronchial carcinoma and 20 patients with benign lung diseases. There were no significant differences in the activities of drug-metabolizing enzymes in the lung tissue of smokers, regardless of whether they smoked low- or high-tar and -nicotine cigarettes; former smokers; or nonsmokers; nor were there such differences between female and male patients. No significant differences existed between patients with squamous cell carcinoma and those with adenocarcinoma. Impaired detoxification due to decreased activity of glutathione S-transferases, rather induction of benzo()pyrene-metabolizing enzymes, may disturb the delicate balance between the generation and detoxification of reactive metabolites. This impairment may lead to the accumulation of these compounds in the cell.Abbreviations BP benzo()pyrene - ECDE 7-ethoxycoumarin O-deethylase - EH epoxide hydrolase - ERDE 7-ethoxyresorufin O-deethylase - GST glutathione S-transferases - LAK lymphokine-activated killer cells - PAH polycyclic aromatic hydrocarbon - SCC squamous cell carcinoma - T/N tar/nicotine This paper is dedicated to the memory of Prof. E.E. Ohnhaus     

Antiherpetic potential of 6-bromoindirubin-3′-acetoxime (BIO-acetoxime) in human oral epithelial cells

Glycogen synthase kinase 3 (GSK-3) functions in the regulation of glycogen metabolism, in the cell cycle, and in immune responses and is targeted by some viruses to favor the viral life cycle. Inhibition of GSK-3 by 6-bromoindirubin-3′-acetoxime (BIO-acetoxime), a synthetic derivative of a compound from the Mediterranean mollusk Hexaplex trunculus, protects cells from varicella infection. In this study, we examined the effects of BIO-acetoxime against herpes simplex virus-1 (HSV-1) infection in human oral epithelial cells, which represent a natural target cell type. The results revealed that BIO-acetoxime relieves HSV-1-induced cytopathic effects and apoptosis. We also found that BIO-acetoxime reduced viral yields and the expression of different classes of viral proteins. Furthermore, addition of BIO-acetoxime before, simultaneously with or after HSV-1 infection significantly reduced viral yields. Collectively, BIO-acetoxime may suppress viral gene expression and protect oral epithelial cells from HSV-1 infection. These results suggest the possible involvement of GSK-3 in HSV-1 infection.     

Increased expression of peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ in human atherosclerosis

To clarify the mechanism of atherosclerosis development in humans, we studied the mRNA and protein expression of PPAR subtypes in various types of atherosclerotic lesions and their correlation with cell proliferation and macrophage invasion. Human aortas were obtained from 35 autopsied cases, and each sample was divided into halves. One half was used for the analysis of mRNA or protein expression with RT-PCR or Western blotting, respectively. The other was microscopically classified into atheromatous plaque (AP), fatty streak (FS), and diffuse intimal thickening (DIT), and was analyzed immunohistochemically. The mRNA levels of both PPARs increased significantly in atherosclerosis and tended to increase in proportion to the severity of the lesion, and the expression of PPAR-α correlated with that of PPAR-γ in FS and AP. The PPAR-γ protein increased in AP. Monocytes/macrophages, as well as endothelial and smooth muscle cells, expressed the PPAR-γ protein in plaques. This expression in the DIT was noted mainly in macrophages but was not correlated with the density of macrophages, suggesting that only certain macrophages express the PPARs in DIT. Cell proliferation did not correlate with PPARs expression in any lesion type. These findings suggest that PPARs may be associated with atheromatous plaque formation, and that PPAR-γ may be involved in the early stages of human atherosclerosis.     

Characterization of the human β4 nAChR gene and polymorphisms in CHRNA3 and CHRNB4

Most neuronal nicotinic acetylcholine receptors are heteropentamers, composed of α and β subunits. Mice lacking the α3 subunit and mice lacking both the β2 and β4 subunits, but not mice lacking the β2 or β4 subunits alone, have a severe phenotype characterized by megacystis, failure of bladder strips to contract in response to nicotine, widely dilated ocular pupils, growth failure, and perinatal mortality. The deficit in bladder contraction was also found in mice lacking only the β4 subunit, although they did not develop megacystis. The major bladder phenotype resembles the human autosomal recessive disorder of megacystis-microcolon-hypoperistalsis syndrome (MMIHS). Based on the similarity of the mouse and human phenotypes, we initiated mutation analyses in the α3 and β4 genes in MMIHS families. The human gene encoding the β4 subunit was fully characterized, including refinement of its mapping. Analysis of disease families and controls identified numerous genetic variants, including high-frequency polymorphisms in both CHRNA3 and CHRNB4. Although no loss-of-function mutations have been identified to date, these genes remain strong candidates for involvement in MMIHS, because various mutations might be obscured within the complex cluster of genes. Some of the markers presented here are valuable tools for analysis of the role of genetic variation in responses to nicotine and for characterization of various dysautonomic abnormalities. Received: February 19, 2001 / Accepted: March 16, 2001     

Long term human reconstitution and immune aging in NOD-Rag (−)-γ chain (−) mice

Aging of the human immune system is characterized by a gradual loss of immune function and a skewing of hematopoiesis toward the myeloid lineage, a reduction in the lymphocytic lineage, and progressive increases in senescent memory T cells at the expense of naïve T cells. Both the innate and the adaptive branches of the immune system are affected, including neutrophils, macrophages, dendritic cells and lymphocytes. Mice, the most common research model, although inexpensive, do not necessarily reflect the human immune system in terms of its interaction with infectious agents of human origin or environmental factors. This study analyzed whether a human immune system contained within the NOD-Rag (−)-γ chain (−) mouse model could realistically be used to evaluate the development and therapy of aging-related diseases. To that end lightly irradiated NOD-Rag (−)-γ chain (−) mice were injected intra-hepatically on day 1 of life with purified cord blood-derived CD34⁺ stem and progenitor cells. Multiple mice were constructed from each cord blood donor. Mice were analyzed quarterly for age-related changes in the hematopoietic and immune systems, and followed for periods up to 18–24 months post-transplant. Flow cytometric analyses were performed for hematopoietic and immune reconstitution. It was observed that NOD-Rag (−)-γ chain (−) mice could be “humanized” long-term using cord blood stem cells, and that some evidence of immune aging occurred during the life of the mice.     

Epistatic interactions between Fc (GM) and FcγR genes and the host control of human immunodeficiency virus replication

Host genetic factors are thought to contribute to the interindividual differences in the control of human immunodeficiency virus (HIV) replication. The aim of the present investigation was to determine whether genes encoding GM and KM allotypes-genetic markers of immunoglobulin γ and κ chains, respectively-and those encoding Fcγ receptor (FcγR) IIa and IIIa are associated with the host control of HIV replication. A case-control design was employed among HIV-infected subjects, with a group that spontaneously controlled HIV replication ("controllers") as cases (n = 73) and those who did not control replication as controls (n = 100). Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism, direct DNA sequencing, and TaqMan genotyping assays. In Caucasian Americans, certain combinations of FcγR and GM genotypes were differentially distributed between controllers and noncontrollers. Among the noncarriers of the FcγRIIa arginine allele, GM21 noncarriers had over 7-fold greater odds of being controllers than the carriers of this allele (odds ratio [OR] = 7.47). These GM determinants also interacted with FcγRIIIa alleles. Among the carriers of the FcγRIIIa valine allele, GM21 noncarriers had over 3-fold greater odds of being controllers than the carriers of this allele (OR = 3.26). These results demonstrate epistatic interactions of genes on chromosomes 14 (GM) and 1 (FcγR) in influencing the control of HIV replication.     

Pontosubicular apoptosis (“necrosis”) in human neonates with intrauterine growth retardation and placental infarction

In a previous study of 37 autopsied stillbirths with non-dysmorphic intrauterine growth retardation (IUGR), 26 cases were associated with placental infarction, a morphologic marker of uteroplacental insufficiency. Nine of the 26 cases with both IUGR and placental infarction, where archival tissue was available, had grey matter ischaemic lesions that were subsequently identified as pontosubicular necrosis. This lesion is now regarded as a localized form of apoptosis. A further eight third trimester stillbirth cases with both IUGR and placental infarction were ascertained prospectively. Sixteen of these 17 cases showed pontosubicular apoptosis, identified morphologically and verified using activated caspase-3 and TUNEL. Five of the 17 cases showed apoptosis in the frontal or temporal cortex as well. In this current study, pontosubicular apoptosis was strongly associated with IUGR and placental infarction in third trimester stillborns, suggesting that uteroplacental insufficiency leading to chronic fetal hypoxaemia may cause cerebral apoptosis.     

Can interferon‐γ and interleukin‐10 balance be associated with severity of human Leishmania (Viannia) braziliensis infection?

Suitable levels of interferon (IFN)-gamma and interleukin (IL)-10 seem to favour the outcome of cutaneous leishmaniasis (CL), while high IFN-gamma and low IL-10 production are associated with severity of mucosal leishmaniasis (ML). Considering that cytokine balance is important for the maintenance of protective responses in leishmaniasis, our aim was to investigate leishmanial antigens-induced IFN-gamma and IL-10 levels maintained in healed individuals who had different clinical outcomes of Leishmania infection. Thirty-three individuals who recovered from L. braziliensis infection were studied: cured CL (CCL), cured ML (CML), spontaneous healing of CL (SH) or asymptomatic individuals (ASY). Cytokines were quantified by enzyme-linked immunosorbent assay (ELISA) in culture supernatants of L. braziliensis-stimulated peripheral blood mononuclear cells (PBMC). IFN-gamma levels were higher in CML (7593 +/- 5994 pg/ml) in comparison to SH (3163 +/- 1526 pg/ml), ASY (1313 +/- 1048 pg/ml) or CCL (1897 +/- 2087 pg/ml). Moreover, cured ML cases maintained significantly lower production of IL-10 (127 +/- 57.8 pg/ml) in comparison to SH (1373 +/- 244 pg/ml), ASY (734 +/- 233 pg/ml) or CCL (542 +/- 375 pg/ml). Thus, a high IFN-gamma/IL-10 ratio observed in CML can indicate unfavourable cytokine balance. On the other hand, no significant difference in the IFN-gamma/IL-10 ratio was observed when CCL individuals were compared to SH or ASY subjects. In conclusion, even after clinical healing, ML patients maintained a high IFN-gamma/IL-10 secretion profile in response to leishmanial antigens. This finding can explain a delayed down-modulation of exacerbated inflammatory responses, which can be related in turn to the necessity of prolonged therapy in ML management. Conversely, lower IFN-gamma/IL-10 balance observed in CCL, SH and ASY individuals can represent a better-modulated immune response associated with a favourable prognosis.     

IgH (μ-switch and γ-1) region restriction fragment length polymorphism in human narcolepsy

Predisposition to narcolepsy involves genetic factors both in humans and in a canine model of the disorder. In humans, narcolepsy is strongly associated with HLA DR15 and DQB1^*0602. In Dobermans and Labradors, narcolepsy is transmitted as a single autosomal recessive gene with full penetrance (canarc-1). Canine narcolepsy is not linked with DLA, the canine equivalent of HLA, but cosegregates with a DNA segment with high homology with the immunoglobulin heavy-chain (IgH) switch-like region (S). To determine if the IgH locus is involved in genetic predisposition to human narcolepsy, restriction fragment length polymorphisms specific for the IgM and IgG cluster within this locus were studied in sporadic cases of the disease, as well as in five families with two or more affected individuals. Comparisons were made between control populations and both familial and sporadic cases and for patients with and without HLA-DR15 and DQB1^*0602. RFLP analysis at the S and-1 loci, which cover over 200 kb of 14q32.3, indicates that there is no evidence for any association between the IgH region and human narcolepsy.     

Regulation of melanoma cell migration and invasion by laminin-5 and α3β1 integrin (VLA-3)

We evaluated the role of soluble factors produced from epidermal cells in melanoma cell motility by using the Boyden chamber chemoinvasion system. The migration of two melanoma cell lines, A375 and Mewo, was potentiated by conditioned media of A431 epidermoid cells in a concentration-dependent manner. The enhancement of A375 melanoma cell motility induced by the conditioned medium was blocked by antibodies against either α3 or β1 integrin subunit. The motility-stimulating activity was recovered in the same fraction as the α3 integrin-dependent adhesion-promoting activity in a high-molecular-weight (>200 kDa) fraction on Superose 12 gel chromatography, and adsorbed with an anti-laminin-5 antibody. Purified laminin-5 was capable of potentiating melanoma cell migration as measured in either the chemotaxis assay with a soluble form of laminin-5 or the haptotaxis assay with membranes coated with a mixture of laminin-5 and Matrigel. Furthermore, immobilized laminin-5 induced A375 melanoma cells to secrete matrix metalloproteinase-9 (type IV collagenase) into the culture medium. These results strongly suggest that the interaction of laminin-5 produced in the epidermis with α3β1 integrin on melanoma cells is involved in cell migration, invasion, and degradation of extracellular matrix proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

1α,25-dihydroxyvitamin D3 (vitamin D3) catalyzes suppressive activity on human natural regulatory T cells, uniquely modulates cell cycle progression, and augments FOXP3

Human natural regulatory T cells (nTregs) show great promise for therapeutically modulating immune-mediated disease, but remain poorly understood. One explanation under intense scrutiny is how to induce suppressive function in non-nTregs and increase the size of the regulatory population. A second possibility would be to make existing nTregs more effective, like a catalyst raises the specific activity of an enzyme. The latter has been difficult to investigate due to the lack of a robust short-term suppression assay. Using a microassay described herein we demonstrate that nTregs in distinct phases of cell cycle progression exhibit graded degrees of potency. Moreover, we show that physiological concentrations of 1α,25-dihydroxyvitamin D3 (vitamin D3) boosts nTregs function. The enhanced suppressive capacity is likely due to vitamin D3's ability to uniquely modulate cell cycle progression and elevate FOXP3 expression. These data suggest a role for vitamin D3 as a mechanism for catalyzing potency of nTregs.     

Why do female Aedes aegypti (Diptera: Culicidae) feed preferentially and frequently on human blood?

Adult female Aedes aegypti (L.), the vector of dengue and yellow fever viruses, have an affinity for feeding on human blood and a tendency to forego feeding on sugar. This observation challenges two tenets of mosquito biology: (1) mosquitoes imbibe plant carbohydrates for synthesis of energy reserves and blood for reproduction and (2) egg production is reduced when mosquitoes feed on human blood compared with blood from other species. Sub-optimal amounts of the amino acid isoleucine in human blood (particularly free isoleucine in plasma) are thought to be responsible for lowered egg production when human blood is ingested. We tested the hypothesis that feeding on human blood is associated with a selective advantage for Ae. aegypti and is an underlying reason for this mosquito's intimate and epidemiologically important relationship with human beings. Our five experiments examined the effects of different isoleucine concentrations on accumulated energy reserves, frequency of host contact, survival, and egg production. When mosquitoes imbibed blood meals over a 7- to 10-d period and were not fed sugar, increased isoleucine concentration decreased energy reserves and did not increase egg production. Aedes aegypti took smaller but more frequent blood meals when feeding on a low-isoleucine human host daily compared with a high-isoleucine mouse host. Previous reports that isoleucine enhances egg production were confirmed only when females were fed sugar, an unusual behavior for most domestic Ae. aegypti populations. Females fed human blood and water had greater age-specific survival (l(x)), reproductive output (m(x)), and cumulative net replacement (R0) than cohorts fed human blood plus sugar or isoleucine-rich mouse blood with or without access to sugar. The unique isoleucine concentration of human blood is associated with Ae. aegypti's unusual propensity to feed preferentially and frequently on humans--a behavior that increases this mosquito's fitness, synthesis of energy reserves, and contact with human hosts, making it an especially effective disseminator of human pathogens.