Increased sensitivity to broadly neutralizing antibodies of end-stage disease R5 HIV-1 correlates with evolution in Env glycosylation and charge

Background

Induction of broadly neutralizing antibodies, such as the monoclonal antibodies IgGb12, 2F5 and 2G12, is the objective of most antibody-based HIV-1 vaccine undertakings. However, despite the relative conserved nature of epitopes targeted by these antibodies, mechanisms underlying the sensitivity of circulating HIV-1 variants to broadly neutralizing antibodies are not fully understood. Here we have studied sensitivity to broadly neutralizing antibodies of HIV-1 variants that emerge during disease progression in relation to molecular alterations in the viral envelope glycoproteins (Env), using a panel of primary R5 HIV-1 isolates sequentially obtained before and after AIDS onset.

Principal Findings

HIV-1 R5 isolates obtained at end-stage disease, after AIDS onset, were found to be more sensitive to neutralization by TriMab, an equimolar mix of the IgGb12, 2F5 and 2G12 antibodies, than R5 isolates from the chronic phase. The increased sensitivity correlated with low CD4⁺ T cell count at time of virus isolation and augmented viral infectivity. Subsequent sequence analysis of multiple env clones derived from the R5 HIV-1 isolates revealed that, concomitant with increased TriMab neutralization sensitivity, end-stage R5 variants displayed envelope glycoproteins (Envs) with reduced numbers of potential N-linked glycosylation sites (PNGS), in addition to increased positive surface charge. These molecular changes in Env also correlated to sensitivity to neutralization by the individual 2G12 monoclonal antibody (mAb). Furthermore, results from molecular modeling suggested that the PNGS lost at end-stage disease locate in the proximity to the 2G12 epitope.

Conclusions

Our study suggests that R5 HIV-1 variants with increased sensitivity to broadly neutralizing antibodies, including the 2G12 mAb, may emerge in an opportunistic manner during severe immunodeficiency as a consequence of adaptive molecular Env changes, including loss of glycosylation and gain of positive charge.     

An anti-HIV-1 V3 loop antibody fully protects cross-clade and elicits T-cell immunity in macaques mucosally challenged with an R5 clade C SHIV

Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization-sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high-dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient "Hit and Run" infection or cross priming via Ag-Ab-mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target.     

Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

Niemann-Pick C (NPC) disease is due to loss of NPC1 or NPC2 protein function that is required for unesterified cholesterol transport from the endosomal/lysosomal compartment. Though lung involvement is a recognized characteristic of Niemann-Pick type C disease, the pathological features are not well understood. We investigated components of the surfactant system in both NPC1 mutant mice and felines and in NPC2 mutant mice near the end of their expected life span. Histological analysis of the NPC mutant mice demonstrated thickened septae and foamy macrophages/leukocytes. At the level of electron microscopy, NPC1-mutant type II cells had uncharacteristically larger lamellar bodies (LB, mean area 2-fold larger), while NPC2-mutant cells had predominantly smaller lamellar bodies (mean area 50% of normal) than wild type. Bronchoalveolar lavage from NPC1 and NPC2 mutant mice had an approx. 4-fold and 2.5-fold enrichment in phospholipid, respectively, and an approx. 9-fold and 35-fold enrichment in cholesterol, consistent with alveolar lipidosis. Phospholipid and cholesterol also were elevated in type II cell LBs and lung tissue while phospholipid degradation was reduced. Enrichment of surfactant protein-A in the lung and surfactant of the mutant mice was found. Immunocytochemical results showed that cholesterol accumulated in the LBs of the type II cells isolated from the affected mice. Alveolar macrophages from the NPC1 and NPC2 mutant mice were enlarged compared to those from wild type mice and were enriched in phospholipid and cholesterol. Pulmonary features of NPC1 mutant felines reflected the disease described in NPC1 mutant mice. Thus, with the exception of lamellar body size, the lung phenotype seen in the NPC1 and NPC2 mutant mice were similar. The lack of NPC1 and NPC2 proteins resulted in a disruption of the type II cell surfactant system contributing to pulmonary abnormalities.     

ORIGINAL ARTICLE: AIDS and optic neuritis in a rhesus monkey infected with the R5 clade C SHIV‐1157ipd3N4

A Chinese rhesus macaque infected with the pathogenic CCR5‐tropic clade C simian‐human immunodeficiency virus, SHIV‐1157ipd3N4, had persistent viremia, depletion of CD4⁺ T cells to <200 cells/μl, opportunistic infections, coagulopathy, and gradual development of bilateral blindness. MRI revealed marked thickening of both optic nerves. Histopathological evaluation showed diffuse cellular infiltration at necropsy and a focus of SHIV‐infected cells. This is the first report of CNS pathology following chronic infection with an obligate R5 SHIV.     

Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A

To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (Δ40) or 25-aa (Δ25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. Δ40 conferred efficient growth characteristics to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log(10) focus-forming units (FFU)/ml. 2a(J6) with Δ40 or Δ25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFPΔ40 acquired various deletions in EGFP, while 2a(J6)Δ40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5AΔ40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those of the nontagged recombinants, with peak infectivity titers of 4 to 5 log(10) FFU/ml. RLuc recombinants showed slightly less efficient growth characteristics, with peak infectivity titers up to 10-fold lower. Overall, the EGFP and RLuc recombinants were genetically stable after one viral passage. The usefulness of these reporter viruses for high-throughput fluorescence- and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN-alfa-2b) and that genotype 1 to 7 viruses all responded to treatment with p7 ion channel inhibitors.     

Liraglutide Achieves A1C Targets More often than Sitagliptin or Exenatide when Added to Metformin in Patients with Type 2 Diabetes and a Baseline A1C <8.0%

ObjectiveCompare the safety and efficacy of liraglutide to that of sitagliptin or exenatide as add-on to metformin in patients with type 2 diabetes (T2D) and glycated hemoglobin (A1C) <8.0%.MethodsPost hoc analysis of 26-week data from liraglutide 1.8 mg once daily (OD) versus exenatide 10 μg twice daily (LEAD-6) and liraglutide 1.8 mg OD versus sitagliptin 100 mg OD (LIRA-DPP-4); only patients treated as add-on to metformin with baseline A1C <8.0% were included. Efficacy analysis was performed on the intention-to-treat population with missing values imputed by last observation carried forward.ResultsMore patients achieved A1C targets (<7.0% and ≤6.5%) with liraglutide versus exenatide or sitagliptin; the difference was greatest for A1C ≤6.5% (LEAD-6: 65% versus 35%; odds ratio [OR]=3.37, 95% confidence interval [CI]: 1.31-8.63; P = .01 or LIRA-DPP-4: 53% versus 19%; OR = 4.78, 95% CI 2.10 to 10.87; P = .0002). Significantly more patients achieved a composite endpoint of A1C <7.0% with no weight gain or hypoglycemia with liraglutide compared with exenatide (78% versus 42%; OR = 4.99, 95% CI: 1.77 to 14.04; P = .0023) or sitagliptin (61% versus 21%; OR = 5.95, 95% CI: 2.66 to 13.29; P<.0001). All treatments were well tolerated, there was no major hypoglycemia and few patients (8 to 10%) experienced minor hypoglycemia.ConclusionWhen added to metformin in patients with an A1C <8.0%, more patients using liraglutide 1.8 mg reached A1C targets than with exenatide or sitagliptin. Sitagliptin had particularly low efficacy in this analysis. These data support the use of liraglutide 1.8 mg as a safe and effective alternative to sitagliptin or exenatide following metformin failure in patients with an A1C <8.0%. (Endocr Pract. 2013;19:64-72)     

Radiation-induced genetic instability: no association with changes in radiosensitivity or cell cycle checkpoints in C3H 10T1/2 mouse fibroblasts

We investigated various phenotypic characteristics of radiation-induced morphologically transformed C3H 10T1/2 mouse fibroblasts. The cells were treated with 8 Gy x-rays, and type II/III foci were isolated. Cell lines were developed from these foci, and subsequently clones were established from these focal lines. The clones were examined for DNA content, radiosensitivity and inducible cell cycle arrests. Besides the morphological changes associated with the transformed state, the major difference between the isolated focal lines or derived clones and the parental C3H 10T1/2 line was one of ploidy. The transformed cells often displayed aneuploid and multiple polyploid populations. No change in the radiosensitivity of the transformed cells was observed. Furthermore, the two major radiation- and staurosporine-induced G1 and G2 cell cycle arrests observed in the parental cell line were also observed in the morphological transformants, suggesting that checkpoint function was normal. Received: 15 May 1997 / Accepted in revised form: 21 October 1997     

AT2R − 1332 G:A polymorphism and its interaction with AT1R 1166 A:C,ACE I/D and MMP-9 − 1562 C:T polymorphisms: Risk factors for susceptibility to preeclampsia

The possible association of angiotensin type 2 receptor (AT2R) − 1332 G:A polymorphism with susceptibility to preeclampsia was studied in 252 women consisted of 155 women with preeclampsia and 97 healthy pregnant women. Also, the interaction of this polymorphism with angiotensin type 1 receptor (AT1R) 1166 A:C, angiotensin converting enzyme insertion/deletion (ACE I/D) and also with matrix metalloproteinase-9 (MMP-9) − 1562 C:T polymorphism was investigated. The AT2R − 1332 G:A polymorphism was detected using PCR–RFLP method. Significantly higher frequencies of GG+GA genotype and G allele of AT2R were observed in mild (80.2%, p = 0.003 and 47.5%, p = 0.012, respectively) and severe (77.8%, p = 0.034 and 48.1%, p = 0.026, respectively) preeclampsia compared to controls (60.8% and 35.1%, respectively). The presence of G allele was associated with 1.69-fold increased risk of preeclampsia (p = 0.005). In severe preeclamptic women, systolic and diastolic blood pressures in the presence of GG+GA genotype were significantly higher compared to those in the presence of AA genotype. The concomitant presence of both alleles of AT2R G and AT1R C was associated with 1.3 times increased risk of mild preeclampsia (p = 0.03). There was an interaction between AT2R G and ACE D alleles that significantly increased the risk of mild and severe preeclampsia by 1.38- and 1.3-fold, respectively. Also, interaction between MMP-9 T and AT2R G alleles increased the risk of severe preeclampsia 1.39-fold (p = 0.028). Our study demonstrated that the G allele of AT2R − 1332 G:A polymorphism is associated with an increased risk of preeclampsia. Also, epistatic interaction of G allele and each allele of the AT1R C, ACE D and MMP-9 T was associated with the risk of preeclampsia. Our findings suggest that the renin–angiotensin system (RAS) variants and gene–gene interactions affect the risk of preeclampsia.     

Activation of the fifth and sixth component of the complement system: similarities between C5b6 and C(56)a with respect to lytic enhancement by cell-bound C3b or A2C, and species preferences of target cell

Brief shift of purified C5 and C6 at 0 degrees C to pH 6.4, followed by immediate neutralization, results in the generation of a factor, designated C(56)a, that lyses erythrocytes together with C7, C8, and C9. We compared C(56)a and C5b6 generated by an alternative-pathway convertase, with regard to their action on different target cells. We found tht C(56)a is similar to C5b6 in the following properties: 1) Together with C7, C(56)a forms a stable intermediate on either sheep or guinea pig erythrocytes. 2) Membrane-bound C3b, or A2C incorporated in the membrane, enhances lysis by C(56)a-9, as well as lysis by C5b6-9. We also found that the lysis of EC(56)a7 or EC5b67 intermediates by C8 and C9 depends on the species of the erythrocytes and the species of C8 and C9. Thus, lysis of sheep erythrocytes is more efficient with guinea pig C8 and C9 than with human C8 and C9. In the case of guinea pig erythrocytes, this relationship is reversed, i.e., these cells lyse more efficiently when human C8 and C9 are used. Enhancement of lysis by membrane-bound C3b or A2C does not abrogate this species incompatibility pattern.     

Saprophytic and pathogenic behaviour of R. solani AG2-1 (ZG-5) in a soil amended with Diplotaxis tenuifolia or Brassica nigra manures and incubated at different temperatures and soil water content

The Brassicaceae species Diplotaxis tenuifolia and Brassica nigra contain high concentrations of glucosinolates, the precursors of isothiocyanates (ITCs) that can have biofumigation effects in amended soils. In a laboratory experiment, incorporation of these plants as green manures into soil was expected to suppress Rhizoctonia solani AG2-1 (ZG5), the causal agent of damping-off in canola (Brassica napus). The manures were incorporated at 1 (1% w/w) or 5 (5% w/w) g fresh material per 100 g dry soil and incubated for 6 months at 10, 20, or 30°C and at soil water contents of 10%, 40%, or 70% of water holding capacity. R. solani survived for up to 6 months as a saprophyte in un-amended soil at all soil water contents and at 10 and 20°C. A temperature of 30°C suppressed R. solani below the level of detection in all treatments after one week. At 1% concentration, the green manures increased the colonisation of the soil by R. solani, which caused severe damping-off of canola subsequently sown in this soil treatment. Soil amendments at 1% temporarily increased soil microbial activity. The addition of B. nigra or D. tenuifolia green manure at 5% concentration suppressed the saprophytic growth of R. solani incubated at 10 or 20°C over all soil water contents and significantly increased the microbial activity at all soil temperatures and water contents. Canola sown into these pots did not succumb to damping-off. The efficiency of hydrolysis of glucosinolates in the 5% treatment in the first week of incubation ranged from 1.6% for 2-propenyl ITC, extracted from soil containing tissues of B. nigra, to 3.4% for 3-butenyl ITC extracted from soil containing tissues of D. tenuifolia. 2-propenyl ITC could not be detected after 7 d of incubation. In the longer term (weeks to months), the increase of microbial activity, caused by adding green manures at 5%, or volatiles from the green manures, most likely played a dominant role in suppressing R. solani. The impact of ITCs, if any, appears to be short-term (days). Responsible Editor: Peter A. H. Bakker     

Susceptibility to postnatal growth retardation induced by 5-AZA-2'-deoxycytidine in utero: gender specificity and correlation with reduced insulin-like growth factor 1

The DNA demethylating agent 5-AZA-2'-deoxyxytidine (5-AZA-CdR) alters gene expression in mice exposed during developmental stages and causes malformations and growth suppression. The aim of this study was to determine if 5-AZA-CdR-induced growth retardation is associated with alterations in energy metabolism or in serum IGF-1 levels. Mice were exposed in utero to 5-AZA-CdR at gestation day 10. At postnatal day 21, exposed pups were weaned and body weights recorded. At 3 months of age, reproductive capacity was studied. At 5 months old, after body weight was recorded mice were killed and serum was collected to determine serum glucose, corticosterone, and IGF-1 levels. The body weights of both treated males and females were reduced at weaning compared with controls, but by 5 months of age, only the male body weight was affected. Reproductive capacity of males and females was reduced with males being more affected. Levels of corticosterone and glucose were not altered. Serum IGF-1 levels were lower in males exposed in utero to 5-AZA-CdR when compared to controls, but not in females, and correlated significantly with body weights. Our data suggest that the decreased levels of IGF-1 associated with the treatment could be the cause of the observed growth retardation in the in utero-exposed mice. A gender dimorphic effect, where males are more affected, is evident.     

BRCA2 in American families with four or more cases of breast or ovarian cancer: recurrent and novel mutations, variable expression, penetrance, and the possibility of families whose cancer is not attributable to BRCA1 or BRCA2.

In order to evaluate the role of inherited BRCA2 mutations in American families--particularly the appearance in America of European founder mutations--the BRCA2 coding sequence, 5' UTR, and 3' UTR were screened in 22 Caucasian American kindreds with four or more cases of breast or ovarian cancer. Six mutations were found that cause a premature-termination codon; four of them have been reported elsewhere, and two are novel. In the four families with previously seen mutations, the distinct lineages at high risk of cancer were of Dutch, German, Irish, and Ashkenazi Jewish ancestry; mutations in Europe reflect these ancestries. The families with novel mutations were Puerto Rican Hispanic (exon 9 deletion 995delCAAAT) and Ashkenazi Jewish (exon 11 deletion 6425delTT). Among female BRCA2-mutation carriers, risks of breast cancer were 32% by age 50 years, 67% by age 70 years, and 80% by age 90 years, yielding a lifetime risk similar to that for BRCA1 but an older distribution of ages at onset. BRCA2 families also included multiple cases of cancers of the male breast (six cases), ovary (three cases), fallopian tube (two cases), pancreas (three cases), bladder (two cases), and prostate (two cases). Among 17 Ashkenazi Jewish families with four or more breast or ovarian cancers, 9 families (including 3 with ovarian cancer and 1 with male breast cancer) carried none of the three ancient mutations in BRCA1 or BRCA2. To date, both BRCA2 and BRCA1 have been screened by SSCA, supplemented by the protein-truncation test, in 48 families with four or more breast or ovarian cancers. Mutations have been detected in BRCA1 in 33 families, in BRCA2 in 6 families, and in neither gene in 9 families, suggesting both the probable cryptic nature of some mutations and the likelihood of at least one other BRCA gene.     

Switching from premixed insulin to glargine-based insulin regimen improves glycaemic control in patients with type 1 or type 2 diabetes: a retrospective primary care-based analysis

Background

Inflammation contributes to cardiovascular complications in type 2 diabetes, which are often characterized by microvascular alterations. We investigated whether myeloid-related protein 8/14 complex (MRP8/14) secreted by transmigrating monocytes and granulocytes may represent a biomarker for microvascular alterations in patients with type 2 diabetes and nephropathy.

Methods

MRP8/14 was measured in 43 patients with type 2 diabetes and nephropathy. Additionally, the inflammatory markers Interleukin-6 (IL-6), Tumor necrosis factor-α (TNF-α) and C-reactive protein (CRP) were quantified. To detect microvascular alterations proteinuria and retinal vessel caliber were used as classical and novel marker, respectively. Proteinuria was quantified by protein-creatinine ratio (PCR); retinal vessel caliber was quantified after retina photography on digitalized retina pictures.

Results

MRP8/14 was positively associated with inflammation (r = 0.57), proteinuria (r = 0.40) and retinal arterial caliber (r = 0.48). Type 2 diabetic patients with MRP8/14 values above the median of 5.8 μg/ml demonstrated higher proteinuria and larger retinal artery caliber than patients with MRP8/14 values below the median (logPCR: -0.51 ± 0.52 versus -0.96 ± 0.46, P < 0.01; retinal artery lumen (μm): 178.3 ± 14.1 versus 162.7 ± 14.9 P < 0.01). Both groups did not differ with regard to metabolic factors and blood pressure. MRP8/14 was an independent predictor of retinal artery caliber in multivariate stepwise regression analysis (β = 0.607) and was positively associated with IL-6 (r = 0.57, P < 0.001) and TNF-α (r = 0.36, P < 0.05).

Conclusion

MRP8/14 – a marker for transendothelial migration – describes not only the state of inflammation in diabetic nephropathy, but additionally the degree of microvascular alterations in the glomerular and retinal bed. Therefore, MRP8/14 may be a potentially selective novel biomarker for microcirculatory defects in diabetic nephropathy.     

Different mechanisms are involved in intracellular calcium increase by insulin-like growth factors 1 and 2 in articular chondrocytes: Voltage-gated calcium channels,and/or phospholipase C coupled to a pertussis-sensitive G-protein

This study describes the mechanisms involved in the IGF-1 and IGF-2-induced increases in intracellular calcium concentration [Ca²⁺]i in cultured chondrocytes and the involvement of type 1 IGF receptors. It shows that IGF-1, IGF-2, and insulin increased the cytosolic free calcium concentration [Ca²⁺]i in a dose-dependent manner, with a plateau from 25 to 100 ng/ml for both IGF-1 and IGF-2 and from 1 to 2 μg/ml for insulin. The effect of IGF-1 was twice as great as the one of IGF-2, and the effect of insulin was 40% lower than IGF-1 effect. Two different mechanisms are involved in the intracellular [Ca²⁺]i increase. 1) IGF-1 and insulin but not IGF-2 involved a Ca²⁺ influx through voltage-gated calcium channels: pretreatment of the cells by EGTA and verapamil diminished the IGF-1 or insulin-induced[Ca²⁺]i but did not block the effect of IGF-2.2)IGF-1, IGF-2, and insulin also induced a Ca²⁺ mobilization from the endoplasmic reticulum: phospholipase C (PLC) inhihitors, neomycin, or U-73122 partially blocked the intracellular [Ca²⁺]i increase induced by IGF-1 and insulin and totally inhibited the effect of IGF-2. This Ca²⁺ mobilization was pertussis toxin (PTX) dependent, suggesting an activation of a PLC coupled to a PTX-sensitive G-protein. Lastly, preincubation of the cells with IGF₁ receptor antibodies diminished the IGF-1-induced Ca²⁺ spike and totally abolished the Ca²⁺ influx, but did not modify the effect of IGF-2. These results suggest that IGF-1 action on Ca²⁺ influx involves the IGF₁ receptor, while part of IGF-1 and all of IGF-2 Ca²⁺ mobilization do not implicate this receptor. J. Cell. Biochem. 64:414–422. © 1997 Wiley-Liss, Inc.     

Participation of Syndecan 2 in the Induction of Stress Fiber Formation in Cooperation with Integrin α5β1: Structural Characteristics of Heparan Sulfate Chains with Avidity to COOH-Terminal Heparin-Binding Domain of Fibronectin

The present study provides direct evidence that syndecan 2 participates selectively in the induction of stress fiber formation in cooperation with integrin α5β1 through specific binding of its heparan sulfate side chains to the fibronectin substrate. Our previous study with Lewis lung carcinoma-derived P29 cells demonstrated that the cell surface heparan sulfate proteoglycan, which binds to fibronectin, is syndecan 2 (N. Itano et al., 1996, Biochem. J. 315, 925–930). We here report that in vitro treatment of the cells by antisense oligonucleotide for syndecan 2 resulted in a failure to form stress fibers on fibronectin substrate in association with specific suppression of its cell surface expression. Instead, localization of actin filaments in the cytoplasmic cortex occurred. A similar response of the cells was observed when the cells were treated to eliminate functions of cell surface heparan sulfates, including exogenous addition of heparin and pretreatment with anti-heparan sulfate antibody, F58-10E4, and with proteinase-free heparitinase I. Size- and structure-defined oligosaccharides prepared from heparin and chemically modified heparins were utilized as competitive inhibitors to examine the structural characteristics of the cell surface heparan sulfates involved in organization of the actin cytoskeleton. Their affinity chromatography on a column linked with a recombinant H-271 peptide containing a C-terminal heparin-binding domain of fibronectin demonstrated that 2-O-sulfated iduronates were essential for the binding. Inhibition studies revealed that a heparin-derived dodecasaccharide sample enriched with an IdoA(2OS)–GlcNS(6OS) disaccharide completely blocked binding of the syndecan 2 ectodomain to immobilized H-271 peptide. Finally, the dodecasaccharide sample was shown to inhibit stress fiber formation, triggered by adhesion of P29 cells to a CH-271 polypeptide consisting of both the RGD cell-binding and the C-terminal heparin-binding domains of fibronectin in a fused form. All these results consistently suggest that syndecan 2 proteoglycan interacts with the C-terminal heparin-binding domain of fibronectin at the highly sulfated cluster(s), such as [IdoA(2OS)–GlcNS(6OS)]₆ present in its heparan sulfate chains, to result in the induction of stress fiber formation in cooperation with integrin α5β1.