kD In Size Research Articles

Staphyloccocus aureus biofilm, in absence of planktonic bacteria, produces factors that activate counterbalancing inflammatory and immune-suppressive genes in human monocytes.

Staphyloccocus aureus (S. aureus) is a major bacterial pathogen in orthopedic periprosthetic joint infection (PJI). S. aureus forms biofilms that promote persistent infection by shielding bacteria from immune cells and inducing an antibiotic-tolerant metabolic state. We developed an in vitro system to study S. aureus biofilm interactions with primary human monocytes in the absence of planktonic bacteria. In line with previous in vivo data, S. aureus biofilm induced expression of inflammatory genes such as TNF and IL1B, and their anti-inflammatory counter-regulator IL10. S. aureus biofilm also activated expression of PD-1 ligands, and IL-1RA, molecules that have the potential to suppress T cell function or differentiation of protective Th17 cells. Gene induction did not require monocyte:biofilm contact and was mediated by a soluble factor(s) produced by biofilm-encased bacteria that was heat resistant and >3 kD in size. Activation of suppressive genes by biofilm was sensitive to suppression by Jak kinase inhibition. These results support an evolving paradigm that biofilm plays an active role in modulating immune responses, and suggest this occurs via production of a soluble vita-pathogen-associated molecular pattern, a molecule that signals microbial viability. Induction of T cell suppressive genes by S. aureus biofilm provides insights into mechanisms that can suppress T cell immunity in PJI.

Identification of a Novel Anti-HIV-1 Protein from Momordica balsamina Leaf Extract.

Our lab investigates the anti-HIV-1 activity in Momordica balsamina (M. balsamina) leaf extract. Traditional Senegalese healers have used M. balsamina leaf extract as a part of a plant-based treatment for HIV/AIDS infections. Our overall goal is to define and validate the scientific basis for using M. balsamina leaf extract as a part of the traditional Senegalese treatment. As an initial characterization of this extract, we used activity-guided fractionation to determine the active ingredient's solubility and relative size. We found that M. balsamina leaf extract inhibits HIV-1 infection by >50% at concentrations of 0.02 mg/mL and above and is not toxic over its inhibitory range (0-0.5 mg/mL). We observed significantly more antiviral activity in direct water and acetonitrile extractions (p ≤ 0.05). We also observed significantly more antiviral activity in the aqueous phases of ethyl acetate, chloroform, and diethyl ether extractions (p ≤ 0.05). Though most of the antiviral activity partitioned into the aqueous layers, some antiviral activity was present in the organic layers. We show that the active agent in the plant extracts is at least 30 kD in size. Significantly more antiviral activity was retained in 3, 10, and 30 kD molecular weight cutoff filters (p ≤ 0.05). In contrast, most of the antiviral activity passed through the 100 kD filter (p ≤ 0.05). Because the active anti-HIV-1 agent presented as a large, amphiphilic molecule we ran the purified extract on an SDS-page gel. We show that the anti-HIV-1 activity in the leaf extracts is attributed to a 30 kDa protein we call MoMo30. This article describes how MoMo30 was determined to be responsible for its anti-HIV-1 activity.

Hydrogel-backed nanopores in proteomic studies

Accurate identification and quantification of proteins in a solution using nanopores is technologically challenging in part because of the large fraction of missed translocation events due to short event times and limitations of conventional current amplifiers. Previously, we have shown that a nanopore interfaced with PEG(1000)-DMA hydrogel significantly enhances protein residence time inside the nanopore, reducing the number of missed events. (Acharya, ACS Sensors 2020) Following up on this work, we also have demonstrated the sensitivity of the hydrogel-backed nanopores to measure unlabeled proteins as small as 5.5 kD in size and 10 fM in concentration.

Contributions of Nitric Oxide to AHR-Ligand-Mediated Keratinocyte Differentiation.

Activation of the aryl hydrocarbon receptor (AHR) in normal human epidermal keratinocytes (NHEKs) accelerates keratinocyte terminal differentiation through metabolic reprogramming and reactive oxygen species (ROS) production. Of the three NOS isoforms, NOS3 is significantly increased at both the RNA and protein levels by exposure to the very potent and selective ligand of the AHR, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Inhibition of NOS with the chemical N-nitro-l-arginine methyl ester (l-NAME) reversed TCDD-induced cornified envelope formation, an endpoint of terminal differentiation, as well as the expression of filaggrin (FLG), a marker of differentiation. Conversely, exposure to the NO-donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), increased the number of cornified envelopes above control levels and augmented the levels of cornified envelopes formed in response to TCDD treatment and increased the expression of FLG. This indicates that nitric oxide signaling can increase keratinocyte differentiation and that it is involved in the AHR-mediated acceleration of differentiation. As the nitrosylation of cysteines is a mechanism by which NO affects the structure and functions of proteins, the S-nitrosylation biotin switch technique was used to measure protein S-nitrosylation. Activation of the AHR increased the S-nitrosylation of two detected proteins of about 72 and 20 kD in size. These results provide new insights into the role of NO and protein nitrosylation in the process of epithelial cell differentiation, suggesting a role of NOS in metabolic reprogramming and the regulation of epithelial cell fate.

Emerging role of metabolomics in rheumatology

The pursuit for understanding disease pathogenesis, in this age of rapid laboratory diagnostics and fast-paced research, has led scientists worldwide to take recourse in hypothesis-free approaches for molecular diagnosis. Metabolomics is one such powerful tool that explores comprehensibly the metabolic alternations in human diseases. It involves study of small molecules of less than 1kD in size by either LSMS or nuclear magnetic resonance. Unlike genomics, which tells us what may have happened, metabolomics reflects what did happen. The NMR technique has an advantage of analyzing metabolites without sample preparation, thereby diminishing artifacts, is less cumbersome and with the latest database on Metabolome; about 30000 metabolites can be identified. The study of metabolomics for several rheumatic diseases, including rheumatoid arthritis, lupus, osteoarthritis and vasculitis, has revealed distinctive metabolic signatures. Thus, metabolomics is a technique that promises precision medicine with better biomarkers, robust predictors of drug response and of disease outcome, discovery of newer metabolites and pathways in disease pathogenesis, and finally, targeted drug development. This review intends to decipher its relevance in common rheumatic diseases.

Low Molecular Weight Components in Aqueous Echinacea Purpurea Leaf Extract Inhibit Melanoma Cell Growth

Melanoma is a skin cancer associated with high mortality. The three year survival rate from advanced melanoma is between 10-15%. One reason for this high mortality rate is that melanoma cells are resistant to traditional chemotherapeutics. Echinacea is a plant genus native to North America with putative anticancer properties. Here, we examined effects of aqueous Echinacea purpurea leaf extract on the growth of melanoma cells and nontransformed fibroblasts. This aqueous extract reduced B16 mouse melanoma cell growth at concentrations that did not inhibit the growth of nontransformed NIH3T3 fibroblasts, suggesting that the extract had biological specificity against transformed cells. We also examined the effect of different fractions of the extract on melanoma cell growth. These data indicate that components less than 3 kD in size exhibited the greatest inhibitory action on melanoma cell growth. In addition, these data indicated that larger components in the extract ameliorate the ability of these low molecular weight compounds to inhibit melanoma cell growth. Furthermore, Echinacea extract inhibited the growth of v-Src transformed LA25 cells without reducing Src kinase activity. Taken together, these results suggest that aqueous Echinacea purpurea extract contains low molecular weight compounds that preferentially inhibit tumor cell growth in the face of oncogenic tyrosine kinase activity. These data suggest future studies to better define bioactive compounds in Echinacea purpurea and evaluate their therapeutic efficacy in vivo.

Components in aqueous Hibiscus rosa-sinensis flower extract inhibit in vitro melanoma cell growth

Skin cancer is extremely common, and melanoma causes about 80% of skin cancer deaths. In fact, melanoma kills over 50 thousand people around the world each year, and these numbers are rising. Clearly, standard treatments are not effectively treating melanoma, and alternative therapies are needed to address this problem. Hibiscus tea has been noted to have medicinal properties, including anticancer effects. Extracts from Hibiscus have been shown to inhibit the growth of a variety of cancer cells. In particular, recent studies found that polyphenols extracted from Hibiscus sabdariffa by organic solvents can inhibit melanoma cell growth. However, effects of aqueous extracts from Hibiscus rosa-sinesis flowers, which are commonly used to make traditional medicinal beverages, have not been examined on melanoma cells. Here, we report that aqueous H. rosa-sinesis flower extract contains compounds that inhibit melanoma cell growth in a dose dependent manner at concentrations that did not affect the growth of nontransformed cells. In addition, these extracts contain low molecular weight growth inhibitory compounds below 3 kD in size that combine with larger compounds to more effectively inhibit melanoma cell growth. Future work should identify these compounds, and evaluate their potential to prevent and treat melanoma and other cancers.

Thrombospondin Type-1 Domain-Containing 7A in Idiopathic Membranous Nephropathy

BackgroundIdiopathic membranous nephropathy is an autoimmune disease. In approximately 70% of patients, it is associated with autoantibodies against the phospholipase A2 receptor 1 (PLA2R1). Antigenic targets in the remaining patients are unknown.MethodsUsing Western blotting, we screened serum samples from patients with idiopathic membranous nephropathy, patients with other glomerular diseases, and healthy controls for antibodies against human native glomerular proteins. We partially purified a putative new antigen, identified this protein by means of mass spectrometry of digested peptides, and validated the results by analysis of recombinant protein expression, immunoprecipitation, and immunohistochemical analysis.ResultsSerum samples from 6 of 44 patients in a European cohort and 9 of 110 patients in a Boston cohort with anti-PLA2R1–negative idiopathic membranous nephropathy recognized a glomerular protein that was 250 kD in size. None of the serum samples from the 74 patients with idiopathic membranous nephropathy who were seropositive for anti-PLA2R1 antibodies, from the 76 patients with other glomerular diseases, and from the 44 healthy controls reacted against this antigen. Although this newly identified antigen is clearly different from PLA2R1, it shares some biochemical features, such as N-glycosylation, membranous location, and reactivity with serum only under nonreducing conditions. Mass spectrometry identified this antigen as thrombospondin type-1 domain-containing 7A (THSD7A). All reactive serum samples recognized recombinant THSD7A and immunoprecipitated THSD7A from glomerular lysates. Moreover, immunohistochemical analyses of biopsy samples from patients revealed localization of THSD7A to podocytes, and IgG eluted from one of these samples was specific for THSD7A.ConclusionsIn our cohort, 15 of 154 patients with idiopathic membranous nephropathy had circulating autoantibodies to THSD7A but not to PLA2R1, a finding that suggests a distinct subgroup of patients with this condition. (Funded by the French National Center for Scientific Research and others.)

Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton.

Eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme.

Viral degradasome hijacks mitochondria to suppress innate immunity

The balance between the innate immunity of the host and the ability of a pathogen to evade it strongly influences pathogenesis and virulence. The two nonstructural (NS) proteins, NS1 and NS2, of respiratory syncytial virus (RSV) are critically required for RSV virulence. Together, they strongly suppress the type I interferon (IFN)-mediated innate immunity of the host cells by degrading or inhibiting multiple cellular factors required for either IFN induction or response pathways, including RIG-I, IRF3, IRF7, TBK1 and STAT2. Here, we provide evidence for the existence of a large and heterogeneous degradative complex assembled by the NS proteins, which we named "NS-degradasome" (NSD). The NSD is roughly ∼300-750 kD in size, and its degradative activity was enhanced by the addition of purified mitochondria in vitro. Inside the cell, the majority of the NS proteins and the substrates of the NSD translocated to the mitochondria upon RSV infection. Genetic and pharmacological evidence shows that optimal suppression of innate immunity requires mitochondrial MAVS and mitochondrial motility. Together, we propose a novel paradigm in which the mitochondria, known to be important for the innate immune activation of the host, are also important for viral suppression of the innate immunity.

An Accurate Boundary Value Problem Solver Applied to Scattering From Cylinders With Corners

In this paper we consider the classic problems of scattering of waves from perfectly conducting cylinders with piecewise smooth boundaries. The scattering problems are formulated as integral equations and solved using a Nystr\"om scheme where the corners of the cylinders are efficiently handled by a method referred to as Recursively Compressed Inverse Preconditioning (RCIP). This method has been very successful in treating static problems in non-smooth domains and the present paper shows that it works equally well for the Helmholtz equation. In the numerical examples we specialize to scattering of E- and H-waves from a cylinder with one corner. Even at a size kd=1000, where k is the wavenumber and d the diameter, the scheme produces at least 13 digits of accuracy in the electric and magnetic fields everywhere outside the cylinder.

EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT HUMAN CONSENSUS INTERFERON-ALPHA IN Escherichia coli UNDER λPL PROMOTER

Recombinant human consensus interferon-alpha (cIFN-α) was obtained by synthesizing a codon-optimized gene composed of the consensus nucleotides at each position in the human alpha interferon family and expressing it in Escherichia coli. The full cIFN-α gene was synthesized in two steps of assembly and amplification by polymerase chain reaction (PCR) using long (45–50 nucleotides) overlapped primers. The two-step PCR resulted in a DNA band of 504 base pairs (bp) corresponding to the calculated size of the cIFN-α gene. The synthetic gene was cloned into temperature-regulated Power3 expression vector. The ligated Power3-cIFN-α (Power3-cIFNα) plasmid carried the cIFN-α gene under transcriptional regulation of the heat-inducible λPL promoter. This expression system was optimized with respect to heat-shock temperature and time of induction in shake flask cultures. The produced cIFN-α protein was characterized by polyacrylamide gel electrophoresis and immunoassays. The majority of the expressed cIFN-α protein of about 19 kD in size accumulated in the form of inclusion bodies. After refolding and purification utilizing single-step ion-exchange chromatography on DEAE-Sepharose, the yield was 70 mg/L. cIFN-α anti-cancer activity was assayed and compared with the commercially available IFN-α2a.

Expression of recombinant human IFNa-2b/IgG4 Fc fusion protein in a baculovirus insect cell system

To investigate a baculovirus insect cell system for expressing an interferon alpha 2b (IFNa2b)/immunoglobulin G-4 (IgG4) Fc fusion protein, which has long-acting antiviral effects. Human IFNa2b and IgG4 Fc cDNAs were generated by molecular cloning and inserted into a baculovirus shuttle vector, which was then transposed into the DH10 Bac strain to form recombinant Bacmid-IFN/Fc. The Bacmid-IFN/Fc was transfected into High five insect cells, and expression of the IFN/Fc fusion protein was detected by Western blotting and its biological activity was assessed by the cytopathic effect inhibition method. The IFNa2b and IgG4 Fc cDNA fragments were successfully amplified by RT-PCR using human peripheral lymphocytes. After cloning into the baculovirus shuttle vector, pFastBac1, and transforming into DH10 Bac competent cells, screening identified positive clones carrying the recombinant Bacmid-IFN/Fc. A Bacmid-IFN/Fc clone was successfully transfected into the High five insect cells and packaged into the baculovirus for expression of the IFN/Fc fusion protein. Western blotting revealed that the fusion protein expression was specific, and yielded a protein of 45 kD in size. The in vitro antiviral activity of the IFN/Fc fusion protein was 580 IU/mL. A novel IFN/Fc fusion protein was successfully generated using a baculovirus insect cell system, which may prove useful for providing future experimental data for development of a new long-acting interferon to treat chronic viral hepatitis.

The Rice Pentatricopeptide Repeat Protein RF5 Restores Fertility in Hong-Lian Cytoplasmic Male-Sterile Lines via a Complex with the Glycine-Rich Protein GRP162

The cytoplasmic male sterility (CMS) phenotype in plants can be reversed by the action of nuclear-encoded fertility restorer (Rf) genes. The molecular mechanism involved in Rf gene-mediated processing of CMS-associated transcripts is unclear, as are the identities of other proteins that may be involved in the CMS-Rf interaction. In this study, we cloned the restorer gene Rf5 for Hong-Lian CMS in rice and studied its fertility restoration mechanism with respect to the processing of the CMS-associated transcript atp6-orfH79. RF5, a pentatricopeptide repeat (PPR) protein, was unable to bind to this CMS-associated transcript; however, a partner protein of RF5 (GRP162, a Gly-rich protein encoding 162 amino acids) was identified to bind to atp6-orfH79. GRP162 was found to physically interact with RF5 and to bind to atp6-orfH79 via an RNA recognition motif. Furthermore, we found that RF5 and GRP162 are both components of a restoration of fertility complex (RFC) that is 400 to 500 kD in size and can cleave CMS-associated transcripts in vitro. Evidence that a PPR protein interacts directly with a Gly-rich protein to form a subunit of the RFC provides a new perspective on the molecular mechanisms underlying fertility restoration.

Tobacco mitochondrial small heat shock protein NtHSP24.6 adopts a dimeric configuration and has a broad range of substrates

There is a broad range of different small heat shock proteins (sHSPs) that have diverse structural and functional characteristics. To better understand the functional role of mitochondrial sHSP, NtHSP24.6 was expressed in Escherichia coli with a hexahistidine tag and purified. The protein was analyzed by non-denaturing PAGE, chemical cross-linking and size exclusion chromatography and the H6NtHSP24.6 protein was found to form a dimer in solution. The in vitro functional analysis of H6NtHSP24.6 using firefly luciferase and citrate synthase demonstrated that this protein displays typical molecular chaperone activity. When cell lysates of E. coli were heated after the addition of H6NtHSP24.6, a broad range of proteins from 10 to 160 kD in size remained in the soluble state. These results suggest that NtHSP24.6 forms a dimer and can function as a molecular chaperone to protect a diverse range of proteins from thermal aggregation.

Evaluation of SDS-Polyacrylamide gel electrophoresis and immunostrip technique for detection of Tobacco mosaic virus and Cucumber mosaic virus in IRAQ

his study was carried out to evaluate the efficiency of electrophoresis on SDS- poly acrylamide slap gel and immunostrip techniques for detection of Tobacco mosaic virus (TMV, genus Tobamovirus) and Cucumber mosaic virus (CMV, genus Cucumovirus, family Bromoviridae), compared with symptoms on diagnostic plants for the two viruses. The results obtained showed that the two methods were effective. The analysis of samples of purified CMV, total proteins from infected cucumber plants, and extracts from infected plants with or without chlorophyll, by electrophoresis on 10% polyacrylamide slap gel containing 0.1% SDS showed two bands of 24 and 26 kd in size, and absent in samples of total protein or extracts of healthy plants. These two proteins represent the coat protein (CP) of CMV. In addition, one 18 kd protein band appeared on SDS- polyacrylamide gel profile which represent the CP of TMV, when samples of purified virus, total protein of infected plants, and plant extracts with or without chlorophyll were analyzed. This band was absent in similar samples from healthy plants. The test of immunostrip specific for CMV showed positive reaction with extracts from melon, cucumber, winter squash, and zucchini infected plants. Similarly, a positive reaction with immunostrip specific for TMV appeared with extracts from tobacco, tomato infected with TMV. No reaction was obtained with healthy plants extract. These results were similar to those obtained from indicator plants for the two viruses.

Antibodies against Crandell Rees Feline Kidney (CRFK) Cell Line Antigens, α-Enolase, and Annexin A2 in Vaccinated and CRFK Hyperinoculated Cats

Cats inoculated with feline herpesvirus 1, calicivirus, and panleukopenia (FVRCP) vaccines grown on the Crandell Rees feline kidney (CRFK) cell line have been shown to develop anti-CRFK antibodies. The identities of common CRFK antigens are unknown. Cats inoculated with CRFK lysates and FVRCP vaccines will develop autoantibodies measurable by Western blot immunoassay. Antigens associated with these antibodies can be isolated for further study. One CRFK hyperinoculated rabbit, 44 age-matched unvaccinated kittens purchased from a commercial vendor. Commonly recognized CRFK antigens were identified by comparison of Western blot immunoassays using sera from a hyperinoculated rabbit and kittens inoculated with CRFK lysate or 1 of 4 commercially available FVRCP vaccines. Antigens were purified from CRFK lysates and sequenced. Antigen recognition was confirmed by Western blot immunoassay and indirect ELISA for 2 proteins using sera from CRFK and FVRCP inoculated kittens. CRFK antigens 47, 40, and 38 kD in size were identified. Protein isolation and sequencing identified 3 CRFK proteins as alpha-enolase, annexin A2, and macrophage capping protein (MCP). Sera from FVRCP and CRFK inoculated cats were confirmed to recognize annexin A2 and alpha-enolase by Western blot immunoassay and indirect ELISA. This study validated the use of Western blot immunoassay for detection of antibodies against CRFK proteins and identified 3 CRFK antigens. In humans, alpha-enolase antibodies are nephritogenic; alpha-enolase and annexin A2 antibodies have been associated with autoimmune diseases. Further research will be necessary to determine the clinical relevance of these findings.

Plasma From Women With Preeclampsia Inhibits Trophoblast Invasion

To assess whether plasma from women with preeclampsia altered trophoblast invasion, SGHPL-4 extravillous trophoblasts were treated with pooled plasma from women with preeclampsia (PE-P; 10%) or with plasma from healthy pregnant controls (C-P). PE-P significantly inhibited SGHPL-4 invasion through Matrigel-coated transwells (P < .01), reduced mitochondrial dehydrogenase activity (P < .01), and increased apoptosis (P < .05); however, invading cells were no more susceptible to PE-P-induced apoptosis than their static counterparts. C-P did not alter rates of invasion, proliferation, or apoptosis. The bioactivity of PE-P was retained after removal of the 6 most abundant plasma proteins using an immunodepletion column (P < .05). Fractionation of PE-P demonstrated that the reduction in invasion was predominantly mediated by factors >100 kd in size. The authors conclude that plasma from women with preeclampsia contains multiple factors that inhibit invasion. These factors do not specifically target invading cells, but instead may reduce the number of cells available to invade.

Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Glypicans are heparan sulfate proteoglycans that are bound to the cell surface by glycosylphosphatidylinositol. While six members of the glypican family are known in mammals, our study focused on glypican 3 (GPC3). Loss-of-function mutations of GPC3 result in the Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by pre- and postnatal liver and other organ overgrowth. GPC3 is overexpressed in human hepatocellular carcinoma; however, its role in normal liver regeneration and hepatocyte proliferation is unknown. Here we investigated the role of GPC3 in hepatocyte proliferation. GPC3 mRNA and protein levels begin to increase 2 days after hepatectomy with peak expression levels by day 5. In hepatocyte cultures, GPC3 reaches a plateau when hepatocyte proliferation decreases. In vitro studies using Morpholino oligonucleotides showed that blocking GPC3 expression promoted hepatocyte growth. Yeast two-hybrid assays revealed that GPC3 interacts with CD81, a member of the tetraspanin family that is reported to be involved in hepatitis C virus infection and cell proliferation. We found that CD81 levels also increased 2 days after partial hepatectomy and toward the end of regeneration. Immunofluorescence showed that CD81 and GPC3 colocalize by 2 and 6 days after hepatectomy. Co-immunoprecipitation validated the interaction of GPC3 and CD81. Our results indicate that GPC3 may be a negative regulator of liver regeneration and hepatocyte proliferation, and that this regulation may involve CD81.

Renal fibroblasts are sensitive to growth-repressing and matrix-reducing factors from activated lymphocytes.

Various forms of nephropathy accompany interstitial fibrosis with lymphocytic infiltration. To probe the relationship between lymphocyte-derived factors and renal fibroblasts, we studied the effect of culture supernatant from lymphocytes stimulated by concanavalin A (ConASN) on the growth and matrix metabolism of rat kidney fibroblasts. 3H-thymidine incorporation and Northern analysis, respectively, revealed that ConASN repressed cell growth and the mRNA level of collagen type I, but dramatically elevated the steady-state expression of metalloproteinase transin/stromelysin. The growth inhibitor in ConASN was moderately heat-sensitive and less than 5 kD in molecular size, qualities that differed from those of transforming growth factor-beta (TGF-beta), IL-1 beta, IL-6, and tumour necrosis factor-alpha (TNF-alpha). The matrix regulatory factor in ConASN was highly heat-sensitive and more than 30 kD in size. Among several lymphokines tested, TNF-alpha produced the same effects as ConASN on the metabolism of extracellular matrix. We hypothesize that lymphocyte-derived factors have a significant role in the attenuation of renal fibrogenesis, as well as its progression, via inhibiting cell growth and matrix accumulation.