Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance.

We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific beta-tubulin isotype III, result in a spectrum of human nervous system disorders that we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show that the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate that normal TUBB3 is required for axon guidance and maintenance in mammals.

VEGF and IGF Delivered from Alginate Hydrogels Promote Stable Perfusion Recovery in Ischemic Hind Limbs of Aged Mice and Young Rabbits.

Biomaterial-based delivery of angiogenic growth factors restores perfusion more effectively than bolus delivery methods in rodent models of peripheral vascular disease, but the same success has not yet been demonstrated in clinically relevant studies of aged or large animals. These studies explore, in clinically relevant models, a therapeutic angiogenesis strategy for the treatment of peripheral vascular disease that overcomes the challenges encountered in previous clinical trials. Alginate hydrogels providing sustained release of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF) were injected into ischemic hind limbs in middle-aged and old mice, and also in young rabbits, as a test of the scalability of this local growth factor treatment. Spontaneous perfusion recovery diminished with increasing age, and only the combination of VEGF and IGF delivery from gels significantly rescued perfusion in middle-aged (13 months) and old (20 months) mice. In rabbits, the delivery of VEGF alone or in combination with IGF from alginate hydrogels, at a dose 2 orders of magnitude lower than the typical doses used in past rabbit studies, enhanced perfusion recovery when given immediately after surgery, or as a treatment for chronic ischemia. Capillary density measurements and angiographic analysis demonstrated the benefit of gel delivery. These data together suggest that alginate hydrogels providing local delivery of low doses of VEGF and IGF constitute a safe and effective treatment for hind-limb ischemia in clinically relevant animal models, thereby supporting the potential clinical translation of this concept.

N-acetylcysteine reduces disease activity by blocking mammalian target of rapamycin in T cells from systemic lupus erythematosus patients: a randomized, double-blind, placebo-controlled trial.

OBJECTIVE: Systemic lupus erythematosus (SLE) patients exhibit T cell dysfunction, which can be regulated through mitochondrial transmembrane potential (Δψm) and mammalian target of rapamycin (mTOR) by glutathione (GSH). This randomized, double-blind, placebo-controlled study was undertaken to examine the safety, tolerance, and efficacy of the GSH precursor N-acetylcysteine (NAC). METHODS: A total of 36 SLE patients received either daily placebo or 1.2 gm, 2.4 gm, or 4.8 gm of NAC. Disease activity was evaluated monthly by the British Isles Lupus Assessment Group (BILAG) index, the SLE Disease Activity Index (SLEDAI), and the Fatigue Assessment Scale (FAS) before, during, and after a 3-month treatment period. Mitochondrial transmembrane potential and mTOR were assessed by flow cytometry. Forty-two healthy subjects matched to patients for age, sex, and ethnicity were studied as controls. RESULTS: NAC up to 2.4 gm/day was tolerated by all patients, while 33% of those receiving 4.8 gm/day had reversible nausea. Placebo or NAC 1.2 gm/day did not influence disease activity. Considered together, 2.4 gm and 4.8 gm NAC reduced the SLEDAI score after 1 month (P = 0.0007), 2 months (P = 0.0009), 3 months (P = 0.0030), and 4 months (P = 0.0046); the BILAG score after 1 month (P = 0.029) and 3 months (P = 0.009); and the FAS score after 2 months (P = 0.0006) and 3 months (P = 0.005). NAC increased Δψm (P = 0.0001) in all T cells, profoundly reduced mTOR activity (P = 0.0009), enhanced apoptosis (P = 0.0004), reversed expansion of CD4-CD8- T cells (mean ± SEM 1.35 ± 0.12-fold change; P = 0.008), stimulated FoxP3 expression in CD4+CD25+ T cells (P = 0.045), and reduced anti-DNA production (P = 0.049). CONCLUSION: This pilot study suggests that NAC safely improves lupus disease activity by blocking mTOR in T lymphocytes.

Biomaterial vaccines capturing pathogen-associated molecular patterns protect against bacterial infections and septic shock.

Most bacterial vaccines work for a subset of bacterial strains or require the modification of the antigen or isolation of the pathogen before vaccine development. Here we report injectable biomaterial vaccines that trigger potent humoral and T-cell responses to bacterial antigens by recruiting, reprogramming and releasing dendritic cells. The vaccines are assembled from regulatorily approved products and consist of a scaffold with absorbed granulocyte-macrophage colony-stimulating factor and CpG-rich oligonucleotides incorporating superparamagnetic microbeads coated with the broad-spectrum opsonin Fc-mannose-binding lectin for the magnetic capture of pathogen-associated molecular patterns from inactivated bacterial-cell-wall lysates. The vaccines protect mice against skin infection with methicillin-resistant Staphylococcus aureus, mice and pigs against septic shock from a lethal Escherichia coli challenge and, when loaded with pathogen-associated molecular patterns isolated from infected animals, uninfected animals against a challenge with different E. coli serotypes. The strong immunogenicity and low incidence of adverse events, a modular manufacturing process, and the use of components compatible with current good manufacturing practice could make this vaccine technology suitable for responding to bacterial pandemics and biothreats.

A Modular Biomaterial Scaffold-Based Vaccine Elicits Durable Adaptive Immunity to Subunit SARS-CoV-2 Antigens.

The coronavirus disease 2019 (COVID-19) pandemic demonstrates the importance of generating safe and efficacious vaccines that can be rapidly deployed against emerging pathogens. Subunit vaccines are considered among the safest, but proteins used in these typically lack strong immunogenicity, leading to poor immune responses. Here, a biomaterial COVID-19 vaccine based on a mesoporous silica rods (MSRs) platform is described. MSRs loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF), the toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid A (MPLA), and SARS-CoV-2 viral protein antigens slowly release their cargo and form subcutaneous scaffolds that locally recruit and activate antigen-presenting cells (APCs) for the generation of adaptive immunity. MSR-based vaccines generate robust and durable cellular and humoral responses against SARS-CoV-2 antigens, including the poorly immunogenic receptor binding domain (RBD) of the spike (S) protein. Persistent antibodies over the course of 8 months are found in all vaccine configurations tested and robust in vitro viral neutralization is observed both in a prime-boost and a single-dose regimen. These vaccines can be fully formulated ahead of time or stored lyophilized and reconstituted with an antigen mixture moments before injection, which can facilitate its rapid deployment against emerging SARS-CoV-2 variants or new pathogens. Together, the data show a promising COVID-19 vaccine candidate and a generally adaptable vaccine platform against infectious pathogens.

Sclerostin antibody stimulates periodontal regeneration in large alveolar bone defects.

Destruction of the alveolar bone in the jaws can occur due to periodontitis, trauma or following tumor resection. Common reconstructive therapy can include the use of bone grafts with limited predictability and efficacy. Romosozumab, approved by the FDA in 2019, is a humanized sclerostin-neutralizing antibody (Scl-Ab) indicated in postmenopausal women with osteoporosis at high risk for fracture. Preclinical models show that Scl-Ab administration preserves bone volume during periodontal disease, repairs bone defects surrounding dental implants, and reverses alveolar bone loss following extraction socket remodeling. To date, there are no studies evaluating Scl-Ab to repair osseous defects around teeth or to identify the efficacy of locally-delivered Scl-Ab for targeted drug delivery. In this investigation, the use of systemically-delivered versus low dose locally-delivered Scl-Ab via poly(lactic-co-glycolic) acid (PLGA) microspheres (MSs) was compared at experimentally-created alveolar bone defects in rats. Systemic Scl-Ab administration improved bone regeneration and tended to increase cementogenesis measured by histology and microcomputed tomography, while Scl-Ab delivered by MSs did not result in enhancements in bone or cemental repair compared to MSs alone or control. In conclusion, systemic administration of Scl-Ab promotes bone and cemental regeneration while local, low dose delivery did not heal periodontal osseous defects in this study.

T-cell and B-cell signaling biomarkers and treatment targets in lupus.

PURPOSE OF REVIEW: Systemic lupus erythematosus is characterized by the production of antinuclear autoantibodies and dysfunction of T-cells, B-cells, and dendritic cells. Here, we review newly recognized genetic factors and mechanisms that underlie abnormal intracellular signal processing and intercellular communication within the immune system in systemic lupus erythematosus. RECENT FINDINGS: Activation of the mammalian target of rapamycin plays a pivotal role in abnormal activation of T and B-cells in systemic lupus erythematosus. In T-cells, increased production of nitric oxide and mitochondrial hyperpolarization were identified as metabolic checkpoints upstream of mammalian target of rapamycin activation. Mammalian target of rapamycin controls the expression T-cell receptor-associated signaling proteins CD4 and CD3zeta through increased expression of the endosome recycling regulator HRES-1/Rab4 gene, mediates enhanced Ca2+ fluxing and skews the expression of tyrosine kinases both in T and B-cells, and blocks the expression of Foxp3 and the expansion of regulatory T-cells. Mitochondrial hyperpolarization and the resultant ATP depletion predispose T-cells to necrosis, thus promoting the dendritic cell activation, antinuclear autoantibody production, and inflammation. SUMMARY: Mitochondrial hyperpolarization, increased activity of mammalian target of rapamycin and Syk kinases, enhanced receptor recycling and Ca2+ flux have emerged as common T and B-cell biomarkers and targets for treatment in systemic lupus erythematosus.

Macrophage migration inhibitory factor (MIF) as a therapeutic target for rheumatoid arthritis and systemic lupus erythematosus.

Introduction. Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine with upstream regulatory roles in innate and adaptive immunity and is implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Several classes of MIF inhibitors such as small molecule inhibitors and peptide inhibitors are in clinical development. Areas covered. The role of MIF in the pathogenesis of RA and SLE is examined; the authors review the structure, physiology and signaling characteristics of MIF and the related cytokine D-DT/MIF-2. The preclinical and clinical trial data for MIF inhibitors are also reviewed; information was retrieved from PubMed and ClinicalTrials.gov using the keywords MIF, D-DT/MIF-2, CD74, CD44, CXCR2, CXCR4, Jab-1, rheumatoid arthritis, systemic lupus erythematosus, MIF inhibitor, small molecule, anti-MIF, anti-CD74, and peptide inhibitor. Expert opinion. Studies in mice and in humans demonstrate the therapeutic potential of MIF inhibition for RA and SLE. MIF- directed approaches could be particularly efficacious in patients with high expression MIF genetic polymorphisms. In patients with RA and SLE and high expression MIF alleles, targeted MIF inhibition could be a precision medicine approach to treatment. Anti-MIF pharmacotherapies could also be steroid-sparing in patients with chronic glucocorticoid dependence or refractory autoimmune disease.

Combined delivery of VEGF and IGF-1 promotes functional innervation in mice and improves muscle transplantation in rabbits.

Microvascular muscle transfer is the gold standard for reanimation following chronic facial nerve paralysis, however, despite the regenerative capacity of peripheral motor axons, poor reinnervation often results in sub-optimal function. We hypothesized that injection of alginate hydrogels releasing growth factors directly into donor tissue would promote reinnervation, muscle regeneration, and function. A murine model of sciatic nerve ligation and neurorrhaphy was first used to assess the ability of gel delivery of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) to promote functional reinnervation. VEGF + IGF-1 gel delivery to aged mice resulted in prolonged ability to control toe movement, increased toe spreading, and improved static sciatic index score, indicative of improved sciatic nerve and neuromuscular junction function. Further, a 26% increase in muscle fiber area, and 2.8 and 3.0-fold increases in muscle contraction force and velocity, respectively, were found compared to blank alginate in the murine model. This strategy was subsequently tested in a rabbit model of craniofacial gracilis muscle transplantation. Electromyography demonstrated a 71% increase in compound muscle action potential 9 weeks after transplantation following treatment with VEGF + IGF-1 alginate, compared to blank alginate in the rabbit model. Improving functional innervation in transplanted muscle via a hydrogel source of growth factors may enhance the therapeutic outcomes of facial palsy treatments and, more broadly, muscle transplantations.

Measurement of Mitochondrial Mass by Flow Cytometry during Oxidative Stress.

Properly assessing mitochondrial health is crucial to understand their role in disease. MitoTracker green (MTG) and nonylacridine orange (NAO) are fluorescent probes which have been commonly used to assess mitochondrial mass. This is based on the assumption that both MTG and NAO accumulate in mitochondria regardless of the mitochondrial transmembrane potential (ΔΨm). Here, we utilized flow cytometry to evaluate the performance of these probes for assessment of mitochondrial mass relative to forward (FSC) and side scatter (SSC) in human peripheral blood lymphocytes (PBL). In isolated mitochondria, two subpopulations were identified by FSC and SSC measurements which were matched to subpopulations stained by MTG and NAO. The performance of these dyes was examined under oxidative and nitrosative stress induced by rotenone and NOC-18 while N-acetylcysteine (NAC) was employed as an antioxidant. Production of reactive oxygen species (ROS) and ΔΨm were monitored in parallel. With respect to representation of mitochondrial mass, neither MTG nor NAO was affected by ΔΨm. However, MTG showed significant correlation with cytosolic and mitochondrial ROS production and nitrosative stress. Our data suggest that NAO may be more suitable than MTG for assessment of mitochondrial mass by flow cytometry during oxidative stress.

Increased mitochondrial electron transport chain activity at complex I is regulated by N-acetylcysteine in lymphocytes of patients with systemic lupus erythematosus.

AIMS: Systemic lupus erythematosus (SLE) patients' peripheral blood lymphocytes (PBL) show mitochondrial dysfunction and oxidative stress. To determine the electrochemical bases of mitochondrial dysfunction, we measured electron transport chain (ETC) activity and its regulation by N-acetylcysteine (NAC) that reversed glutathione depletion and improved disease activity in SLE. ETC activity was assessed in PBL of 69 SLE patients and 37 healthy donors. Negatively isolated T cells were examined in 7 SLE patients, 11 healthy donors, and 10 nonlupus inflammatory arthritis (IA) donors. RESULTS: O2 consumption (in nmol/ml/min) by lupus PBL was increased at baseline (SLE: 2.492±0.196, control: 2.137±0.153; p=0.027) and with complex IV substrates (SLE: 7.722±0.419, control: 7.006±0.505; p=0.028). SLE PBL consumed more O2 upon in-chamber T-cell activation (p=0.012). After overnight T-cell stimulation, ETC activity of SLE PBL was 2.27-fold increased through complex I (SLE: 1.606±0.273, control: 0.709±0.169; p=0.001) and, to a lesser extent, through complex IV. Likewise, complex I activity was elevated in negatively isolated "untouched" T cells of SLE patients (1.816±0.180) relative to healthy controls (0.917±0.094; p=0.0003) and IA disease controls studied in parallel (1.057±0.199; p=0.0308). NAC diminished O2 consumption through complex I and H2O2 levels both in SLE and in control PBL. INNOVATION: O2 consumption was found to be increased in SLE patients' PBL relative to control subjects evaluated in parallel. ETC complex I is identified as the main source of oxidative stress in SLE. CONCLUSIONS: Lupus PBL exhibit increased O2 consumption through mitochondrial ETC complex I that is inhibited by NAC, which may have therapeutic efficacy through reducing oxidative stress in SLE.

Identification of immune factors regulating antitumor immunity using polymeric vaccines with multiple adjuvants.

The innate cellular and molecular components required to mediate effective vaccination against weak tumor-associated antigens remain unclear. In this study, we used polymeric cancer vaccines incorporating different classes of adjuvants to induce tumor protection, to identify dendritic cell (DC) subsets and cytokines critical to this efficacy. Three-dimensional, porous polymer matrices loaded with tumor lysates and presenting distinct combinations of granulocyte macrophage colony-stimulating factor (GM-CSF) and various Toll-like receptor (TLR) agonists affected 70% to 90% prophylactic tumor protection in B16-F10 melanoma models. In aggressive, therapeutic B16 models, the vaccine systems incorporating GM-CSF in combination with P(I:C) or CpG-ODN induced the complete regression of solid tumors (≤40 mm(2)), resulting in 33% long-term survival. Regression analysis revealed that the numbers of vaccine-resident CD8(+) DCs, plasmacytoid DCs (pDC), along with local interleukin (IL)-12, and granulocyte colony-stimulating factor (G-CSF) concentrations correlated strongly to vaccine efficacy regardless of adjuvant type. Furthermore, vaccine studies in Batf3(-/-) mice revealed that CD8(+) DCs are required to affect tumor protection, as vaccines in these mice were deficient in cytotoxic T lymphocytes priming and IL-12 induction in comparison with wild-type. These studies broadly demonstrate that three-dimensional polymeric vaccines provide a potent platform for prophylactic and therapeutic protection, and can be used as a tool to identify critical components of a desired immune response. Specifically, these results suggest that CD8(+) DCs, pDCs, IL-12, and G-CSF play important roles in priming effective antitumor responses with these vaccines.

Assessment of mitochondrial dysfunction in lymphocytes of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by abnormal activation and cell death signaling within the immune system. Activation, proliferation, or death of cells of the immune system is dependent on controlled reactive oxygen intermediates (ROI) production and ATP synthesis in mitochondria. The mitochondrial transmembrane potential (∆ψ (m)) reflects the energy stored in the electrochemical gradient across the inner mitochondrial membrane which, in turn, is used by F(0)F(1)-ATPase to convert ADP to ATP during oxidative phosphorylation. Mitochondrial hyperpolarization (MHP) and transient ATP depletion represent early and reversible steps in T cell activation and apoptosis. By contrast, T lymphocytes of patients with SLE exhibit elevated ∆ψ (m), i.e., persistent mitochondrial hyperpolarization (MHP), cytoplasmic alkalinization, increased ROI production, as well as diminished levels of intracellular glutathione and ATP. Increased production of nitric oxide has been identified as a cause of MHP and increased mitochondrial biogenesis. Oxidative stress affects signaling through the T cell receptor as well as activity of redox--sensitive caspases. ATP depletion causes diminished activation-induced apoptosis and sensitizes lupus T cells to necrosis. Activation of the mammalian target of rapamycin (mTOR) has recently emerged as a key sensor of MHP and mediator of enhanced Ca(2+) flux in lupus T cells.

Relationship of vaccine efficacy to the kinetics of DC and T-cell responses induced by PLG-based cancer vaccines.

Cancer vaccines are typically formulated for bolus injection and often produce short-lived immunostimulation resulting in poor temporal control over immune cell activation and weak oncolytic activity. One means of overcoming these limitations utilizes immunologically active biomaterial constructs. We previously reported that antigen-laden, macroporous PLG scaffolds induce potent dendritic cell (DC) and cytotoxic T-lymphocyte (CTL) responses via the controlled signaling of inflammatory cytokines, antigen and toll-like receptor agonists. In this study, we describe the kinetics of these responses and illustrate their fundamental relationship to potent tumor rejection when implanted subcutaneously in a mouse B16 model of melanoma. By explanting scaffolds from mice at times ranging from 1-7 d, a seamless relationship was observed between the production of controlled CTL responses, tumor growth and long-term survival in both prophylactic and therapeutic models. Scaffolds must be implanted for > 7 d to augment CTL responses via the prolonged presentation of tumor antigen, and the benefits included a notable regression of established tumors. Host DC infiltration into the porous material persisted for 12 days (peaking at day 5 ~1.4 x 10(6) cells), and a sharp attenuation in DC numbers coincided with peak CD8(+) CTL infiltration at day 12 (~8 x 10(5) cells). Importantly, these PLG systems enhanced DC numbers in the draining lymph node, resulting in increased CD(+) CTL subsets at days 10-16 of vaccination. These results indicate that material systems can finely control innate and adaptive immune cell responses to kill typically untreatable melanoma tumors and provide critical kinetic data for the design of vaccine carriers.

The efficacy of intracranial PLG-based vaccines is dependent on direct implantation into brain tissue.

We previously engineered a macroporous, polymer-based vaccine that initially produces GM-CSF gradients to recruit local dendritic cells and subsequently presents CpG oligonucleotides, and tumor lysate to cell infiltrates to induce immune cell activation and immunity against tumor cells in peripheral tumor models. Here, we demonstrate that this system eradicates established intracranial glioma following implantation into brain tissue, whereas implantation in resection cavities obviates vaccine efficacy. Rats bearing seven-day old, intracranial glioma tumors were treated with PLG vaccines implanted into the tumor bed, resulting in retention of contralateral forelimb function (day 17) that is compromised by tumor formation in control animals, and 90% long-term survival (>100 days). Similar benefits were observed in animals receiving tumor resection plus vaccine implants into the adjacent parenchyma, but direct implantation of PLG vaccines into the resection cavity conferred no benefit. This dissociation of efficacy was likely related to GM-CSF distribution, as implantation of PLG vaccines within brain tissue produced significant GM-CSF gradients for prolonged periods, which was not detected after implantation in resection cavities. These studies demonstrate that PLG vaccine efficacy is correlated to GM-CSF gradient formation, which requires direct implantation into brain tissue, and justify further exploration of this approach for glioma treatment.

Prevention of hepatocarcinogenesis and increased susceptibility to acetaminophen-induced liver failure in transaldolase-deficient mice by N-acetylcysteine.

Although oxidative stress has been implicated in acute acetaminophen-induced liver failure and in chronic liver cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathway has been identified. Recent case reports suggest a link between the pentose phosphate pathway (PPP) enzyme transaldolase (TAL; encoded by TALDO1) and liver failure in children. Here, we show that Taldo1-/- and Taldo1+/- mice spontaneously developed HCC, and Taldo1-/- mice had increased susceptibility to acetaminophen-induced liver failure. Oxidative stress in Taldo1-/- livers was characterized by the accumulation of sedoheptulose 7-phosphate, failure to recycle ribose 5-phosphate for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides. Furthermore, we found evidence of hepatic mitochondrial dysfunction, as indicated by loss of transmembrane potential, diminished mitochondrial mass, and reduced ATP/ADP ratio. Reduced beta-catenin phosphorylation and enhanced c-Jun expression in Taldo1-/- livers reflected adaptation to oxidative stress. Taldo1-/- hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of the potent antioxidant N-acetylcysteine (NAC) prevented acetaminophen-induced liver failure, restored Fas-dependent hepatocyte apoptosis, and blocked hepatocarcinogenesis in Taldo1-/- mice. These data reveal a protective role for the TAL-mediated branch of the PPP against hepatocarcinogenesis and identify NAC as a promising treatment for liver disease in TAL deficiency.

Molecular mimicry and immunomodulation by the HRES-1 endogenous retrovirus in SLE.

Genetic and environmental factors are believed to influence development of systemic lupus erythematosus (SLE). Endogenous retroviruses (ERV) correspond to the integrated proviral form of infectious retroviruses, which are trapped within the genome due to mutations. ERV represent a key molecular link between the host genome and infectious viral particles. ERV-encoded proteins are recognized by antiviral immune responses and become targets of autoreactivity. Alternatively, ERV protein may influence cellular processes and the life cycle of infectious viruses. As examples, the HRES-1 human ERV encodes a 28-kDa nuclear autoantigen and a 24-kDa small GTP-ase, termed HRES-1/Rab4. HRES-1/p28 is a nuclear autoantigen recognized by cross-reactive antiviral antibodies, while HRES-1/Rab4 regulates surface expression of CD4 and the transferrin receptor (TFR) through endosome recycling. Expression of HRES-1/Rab4 is induced by the tat gene of HIV-1, which in turn down-regulates expression of CD4 and susceptibility to re-infection by HIV-1. CD4 and the TFR play essential roles in formation of the immunological synapse (IS) during normal T-cell activation by a cognate MHC class II peptide complex. The key intracellular transducer of T-cell activation, Lck, is brought to the IS via binding to CD4. T-cell receptorzeta (TCRzeta) chain binds to the TFR. Abnormal T-cell responses in SLE have been associated with reduced lck and TCRzeta chain levels. HRES-1 is centrally located on chromosome 1 at q42 relative to lupus-linked microsatellite markers and polymorphic HRES-1 alleles have been linked to the development of SLE. 1q42 is one of the three most common fragile sites in the human genome, and is inducible by DNA demethylation, a known mechanism of retroviral gene activation. Molecular mimicry and immunomodulation by a ERV, such as HRES-1, may contribute to self-reactivity and abnormal T and B-cell functions in SLE.