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CD4 + T cells play a central role in the adaptive immune response through their capacity to activate, support and control other immune cells. Although these cells have become the focus of intense research, a comprehensive understanding of the underlying regulatory networks that orchestrate CD4 + T cell function and activation is still incomplete. Here, we analyzed a large transcriptomic dataset consisting of 48 different human CD4 + T cell conditions. By performing reverse network engineering, we identified six common denominators of CD4 + T cell functionality (CREB1, E2F3, AHR, STAT1, NFAT5 and NFATC3). Moreover, we also analyzed condition-specific genes which led us to the identification of the transcription factor MEOX1 in T reg cells. Expression of MEOX1 was comparable to FOXP3 in T reg cells and can be upregulated by IL-2. Epigenetic analyses revealed a permissive epigenetic landscape for MEOX1 solely in T reg cells. Knockdown of MEOX1 in T reg cells revealed a profound impact on downstream gene expression programs and T reg cell suppressive capacity. These findings in the context of CD4 + T cells contribute to a better understanding of the transcriptional networks and biological mechanisms controlling CD4 + T cell functionality, which opens new avenues for future therapeutic strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Baßler, Schmidleithner, Shakiba, Elmzzahi, Köhne, Floess, Scholz, Ohkura, Sadlon, Klee, Neubauer, Sakaguchi, Barry, Huehn, Bonaguro, Ulas and Beyer.)

Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows chromatin accessibility profiling from low cell input, making it tractable on rare cell populations, such as regulatory T (Treg) cells. However, little is known about the compatibility of the assay with cryopreserved rare cell populations. Here we demonstrate the robustness of an ATAC-seq protocol comparing primary Treg cells recovered from fresh or cryopreserved PBMC samples, in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 cryopreserved Treg cells. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and is particularly beneficial when constrained by limited input material. Overall, we observed a high correlation of accessibility patterns and transcription factor dynamics between fresh and cryopreserved samples. Furthermore, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. We highlight the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories., (© 2023. The Author(s).)

Background: Genome-wide association studies (GWAS) have enabled the discovery of single nucleotide polymorphisms (SNPs) that are significantly associated with many autoimmune diseases including type 1 diabetes (T1D). However, many of the identified variants lie in non-coding regions, limiting the identification of mechanisms that contribute to autoimmune disease progression. To address this problem, we developed a variant filtering workflow called 3DFAACTS-SNP to link genetic variants to target genes in a cell-specific manner. Here, we use 3DFAACTS-SNP to identify candidate SNPs and target genes associated with the loss of immune tolerance in regulatory T cells (Treg) in T1D., Results: Using 3DFAACTS-SNP, we identified from a list of 1228 previously fine-mapped variants, 36 SNPs with plausible Treg-specific mechanisms of action. The integration of cell type-specific chromosome conformation capture data in 3DFAACTS-SNP identified 266 regulatory regions and 47 candidate target genes that interact with these variant-containing regions in Treg cells. We further demonstrated the utility of the workflow by applying it to three other SNP autoimmune datasets, identifying 16 Treg-centric candidate variants and 60 interacting genes. Finally, we demonstrate the broad utility of 3DFAACTS-SNP for functional annotation of all known common (> 10% allele frequency) variants from the Genome Aggregation Database (gnomAD). We identified 9376 candidate variants and 4968 candidate target genes, generating a list of potential sites for future T1D or other autoimmune disease research., Conclusions: We demonstrate that it is possible to further prioritise variants that contribute to T1D based on regulatory function, and illustrate the power of using cell type-specific multi-omics datasets to determine disease mechanisms. Our workflow can be customised to any cell type for which the individual datasets for functional annotation have been generated, giving broad applicability and utility., (© 2022. The Author(s).)

Evaluation of penicillin and oxacillin susceptibility testing was conducted on 200 Staphylococcus lugdunensis isolates. Disc diffusion with penicillin 1 IU (P1, EUCAST) and penicillin 10 IU (P10, CLSI) was compared with nitrocefin discs (Cefinase) and automated broth microdilution (Vitek 2). Oxacillin susceptibility was extrapolated from cefoxitin (FOX; 30 μg) disc diffusion and compared with Vitek 2 results. The reference methods were blaZ and mecA PCR. Penicillin zone diameter and zone edge correlated with blaZ PCR results in all except two P10-susceptible isolates (very major error [VME]) and one P1-resistant isolate (major error [ME]). A total of 148 isolates were blaZ negative, of which 146 and 149 isolates were susceptible by P1 and P10, respectively. A total of 127 were penicillin susceptible by Vitek 2. Vitek 2 overcalled resistance in 21 blaZ -negative, 20 P1-susceptible, and 22 P10-susceptible isolates (Vitek 2 ME rate, 14.2%). Two mecA -positive isolates were oxacillin resistant by FOX disc and Vitek 2 methods (categorical agreement). However, 18 FOX-susceptible mecA -negative isolates tested resistant by Vitek 2. In conclusion, Vitek 2 overestimated penicillin and oxacillin resistance compared with disc diffusion and PCR results. In our study, disc diffusion with zone edge interpretation was more accurate and specific than automated broth microdilution for S. lugdunensis.

Eukaryotic genomes are highly organised within the nucleus of a cell, allowing widely dispersed regulatory elements such as enhancers to interact with gene promoters through physical contacts in three-dimensional space. Recent chromosome conformation capture methodologies such as Hi-C have enabled the analysis of interacting regions of the genome providing a valuable insight into the three-dimensional organisation of the chromatin in the nucleus, including chromosome compartmentalisation and gene expression. Complicating the analysis of Hi-C data, however, is the massive amount of identified interactions, many of which do not directly drive gene function, thus hindering the identification of potentially biologically functional 3D interactions. In this review, we collate and examine the downstream analysis of Hi-C data with particular focus on methods that prioritise potentially functional interactions. We classify three groups of approaches: structural-based discovery methods, e.g. A/B compartments and topologically associated domains, detection of statistically significant chromatin interactions, and the use of epigenomic data integration to narrow down useful interaction information. Careful use of these three approaches is crucial to successfully identifying potentially functional interactions within the genome., (© 2021. The Author(s).)

Background: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated., Methods: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 10 6 cells/mL to 1.67 × 10 6 cells/mL., Results: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 10 6 cells/mL., Conclusion: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.

There has been much interest in the ability of regulatory T cells (Treg) to switch function in vivo , either as a result of genetic risk of disease or in response to environmental and metabolic cues. The relationship between levels of FOXP3 and functional fitness plays a significant part in this plasticity. There is an emerging role for Treg in tissue repair that may be less dependent on FOXP3, and the molecular mechanisms underpinning this are not fully understood. As a result of detailed, high-resolution functional genomics, the gene regulatory networks and key functional mediators of Treg phenotype downstream of FOXP3 have been mapped, enabling a mechanistic insight into Treg function. This transcription factor-driven programming of T-cell function to generate Treg requires the switching on and off of key genes that form part of the Treg gene regulatory network and raises the possibility that this is reversible. It is plausible that subtle shifts in expression levels of specific genes, including transcription factors and non-coding RNAs, change the regulation of the Treg gene network. The subtle skewing of gene expression initiates changes in function, with the potential to promote chronic disease and/or to license appropriate inflammatory responses. In the case of autoimmunity, there is an underlying genetic risk, and the interplay of genetic and environmental cues is complex and impacts gene regulation networks frequently involving promoters and enhancers, the regulatory elements that control gene expression levels and responsiveness. These promoter-enhancer interactions can operate over long distances and are highly cell type specific. In autoimmunity, the genetic risk can result in changes in these enhancer/promoter interactions, and this mainly impacts genes which are expressed in T cells and hence impacts Treg/conventional T-cell (Tconv) function. Genetic risk may cause the subtle alterations to the responsiveness of gene regulatory networks which are controlled by or control FOXP3 and its target genes, and the application of assays of the 3D organization of chromatin, enabling the connection of non-coding regulatory regions to the genes they control, is revealing the direct impact of environmental/metabolic/genetic risk on T-cell function and is providing mechanistic insight into susceptibility to inflammatory and autoimmune conditions., (Copyright © 2020 Brown, Sadlon, Hope, Wong, Wong, Liu, Withers, Brown, Bandara, Gundsambuu, Pederson, Breen, Robertson, Forrest, Beyer and Barry.)

CD4 + T-cell subsets play a major role in the host response to infection, and a healthy immune system requires a fine balance between reactivity and tolerance. This balance is in part maintained by regulatory T cells (Treg), which promote tolerance, and loss of immune tolerance contributes to autoimmunity. As the T cells which drive immunity are diverse, identifying and understanding how these subsets function requires specific biomarkers. From a human CD4 Tconv/Treg cell genome wide analysis we identified peptidase inhibitor 16 (PI16) as a CD4 subset biomarker and we now show detailed analysis of its distribution, phenotype and links to Treg function in type 1 diabetes. To determine the clinical relevance of Pi16 Treg, we analysed PI16 + Treg cells from type 1 diabetes patient samples. We observed that FOXP3 expression levels declined with disease progression, suggesting loss of functional fitness in these Treg cells in Type 1 diabetes, and in particular the rate of loss of FOXP3 expression was greatest in the PI16+ve Treg. We propose that PI16 has utility as a biomarker of functional human Treg subsets and may be useful for tracking loss of immune function in vivo. The ability to stratify at risk patients so that tailored interventions can be applied would open the door to personalised medicine for Type 1 diabetes., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E 2 (PGE 2 ) into the metabolite 15-keto PGE 2 , was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE 2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Regulatory T cells (Treg) are critical for preventing autoimmunity and curtailing responses of conventional effector T cells (Tconv). The reprogramming of T-cell fate and function to generate Treg requires switching on and off of key gene regulatory networks, which may be initiated by a subtle shift in expression levels of specific genes. This can be achieved by intermediary regulatory processes that include microRNA and long noncoding RNA-based regulation of gene expression. There are well-documented microRNA profiles in Treg and Tconv, and these can operate to either reinforce or reduce expression of a specific set of target genes, including FOXP3 itself. This type of feedforward/feedback regulatory loop is normally stable in the steady state, but can alter in response to local cues or genetic risk. This may go some way to explaining T-cell plasticity. In addition, in chronic inflammation or autoimmunity, altered Treg/Tconv function may be influenced by changes in enhancer-promoter interactions, which are highly cell type-specific. These interactions are impacted by genetic risk based on genome-wide association studies and may cause subtle alterations to the gene regulatory networks controlled by or controlling FOXP3 and its target genes. Recent insights into the 3D organisation of chromatin and the mapping of noncoding regulatory regions to the genes they control are shedding new light on the direct impact of genetic risk on T-cell function and susceptibility to inflammatory and autoimmune conditions.

Human metapneumovirus (hMPV), first described in 2001 [1], is responsible for causing serious respiratory illness in young children, the elderly and immunocompromised patients. Four distinct lineages of hMPV have been identified with the original nomenclature for these subgroups (A1, A2, B1 and B2), reported by van den Hoogen et al. [2], utilised by many. An alternate terminology (1A, 1B, 2A and 2B) was also published by Ishiguro et al. in 2004 [3] which has been adopted by others. However, this has caused some confusion in the interpretation of publication results as the terminology is similar yet describes different subtypes. As a result, a number of investigators have made a submission to the International Committee on Taxonomy of Viruses (ICTV, ICTV taxonomic proposal 2012.012V) for the official adoption of the original terminology as an approved nomenclature for hMPV [4]. We welcome this officially approved nomenclature which should provide clarification of these subtypes in future. Therefore to assist with the interpretation of our recently published research in the 2012 special issue of Viruses: Pneumoviruses and Metapneumoviruses entitled "Diversity in Glycosaminoglycan Binding Amongst hMPV G Protein Lineages" [5] we have updated the Figure 3 in this letter (see Figure 1), showing the proposed ICTV terminology compared to the Ishiguro classification (used in our publication). Note that in the original publication the alphanumeric order for the Ishiguro classification was transposed (e.g., 1A was referred to as A1).

We have previously shown that hMPV G protein (B2 lineage) interacts with cellular glycosaminoglycans (GAGs). In this study we examined subtypes A1, A2 and B1 for this interaction. GAG-dependent infectivity of available hMPV strains was demonstrated using GAG-deficient cells and heparin competition. We expressed the G protein ectodomains from all strains and analysed these by heparin affinity chromatography. In contrast to the B2 lineage, neither the A2 or B1 G proteins bound to heparin. Sequence analysis of these strains indicated that although there was some homology with the B2 heparin-binding domains, there were less positively charged residues, providing a likely explanation for the lack of binding. Although sequence analysis did not demonstrate well defined positively charged domains in G protein of the A1 strain, this protein was able to bind heparin, albeit with a lower affinity than G protein of the B2 strain. These results indicate diversity in GAG interactions between G proteins of different lineages and suggest that the GAG-dependency of all strains may be mediated by interaction with an alternative surface protein, most probably the conserved fusion (F) protein. Analysis of both native and recombinant F protein confirmed that F protein binds heparin, supporting this conclusion.

Natural Regulatory T cells (Tregs) are defined by stable expression of the cell surface proteins CD4 and CD25, low surface expression of CD127 and expression of the transcription factor FOXP3. The contribution of Treg to the prevention of autoimmunity and the maintenance of immune homoestasis is the subject of ongoing interest, as alterations in Treg numbers and function are implicated in a wide range of diseases. The in vitro benchmark for determining Treg function is suppression of proliferation of unmatched effector T cells in a mixed lymphocyte reaction (MLR) over a 3-6-day time period. As an alternative to this assay, we show that a 7-h CD154 expression assay is rapid, simple and provides a reliable readout of suppressor function. Using multiple Treg-like cell types including natural (n)Treg, inducible (i)Treg and Treg cell lines, we show that suppression of CD154 expression is a surrogate for suppression of proliferation. We propose this as a suitable alternative to the MLR assay, as it is rapid and may be more amenable to high-throughput screening, analysing large cohorts of clinical samples or assaying transiently suppressive populations.

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.

Recent data indicate that a variety of regulatory molecules active in embryonic development may also play a role in the regulation of early hematopoiesis. Here we report that the human Vent-like homeobox gene VENTX, a putative homolog of the Xenopus xvent2 gene, is a unique regulatory hematopoietic gene that is aberrantly expressed in CD34(+) leukemic stem-cell candidates in human acute myeloid leukemia (AML). Quantitative RT-PCR documented expression of the gene in lineage positive hematopoietic subpopulations, with the highest expression in CD33(+) myeloid cells. Notably, expression levels of VENTX were negligible in normal CD34(+)/CD38(-) or CD34(+) human progenitor cells. In contrast to this, leukemic CD34(+)/CD38(-) cells from AML patients with translocation t(8,21) and normal karyotype displayed aberrantly high expression of VENTX. Gene expression and pathway analysis demonstrated that in normal CD34(+) cells enforced expression of VENTX initiates genes associated with myeloid development and down-regulates genes involved in early lymphoid development. Functional analyses confirmed that aberrant expression of VENTX in normal CD34(+) human progenitor cells perturbs normal hematopoietic development, promoting generation of myeloid cells and impairing generation of lymphoid cells in vitro and in vivo. Stable knockdown of VENTX expression inhibited the proliferation of human AML cell lines. Taken together, these data extend our insights into the function of embryonic mesodermal factors in human postnatal hematopoiesis and indicate a role for VENTX in normal and malignant myelopoiesis.

Manipulation of gene expression is an invaluable tool to study gene function in vitro and in vivo. The application of small inhibitory RNAs to knock down gene expression provides a relatively simple, elegant, but transient approach to study gene function in many cell types as well as in whole animals. Short hairpin structures (shRNAs) are a logical advance as they can be expressed continuously and are hence suitable for stable gene knockdown. Drug-inducible systems have now been developed; however, application of the technology has been hampered by persistent problems with low or transient expression, leakiness or poor inducibility of the short hairpin, and lack of reversibility. We have developed a robust, versatile, single lentiviral vector tool that delivers tightly regulated, fully reversible, doxycycline-responsive knockdown of target genes (FOXP3 and MYB), using single short hairpin RNAs. To demonstrate the capabilities of the vector we targeted FOXP3 because it plays a critical role in the development and function of regulatory T cells. We also targeted MYB because of its essential role in hematopoiesis and implication in breast cancer progression. The versatility of this vector is hence demonstrated by knockdown of distinct genes in two biologically separate systems.

Activation of C3 to C3b signals the start of the alternative complement pathway. The C-terminal short complement regulator (SCR)-20 domain of factor H (FH), the major serum regulator of C3b, possesses a binding site for C3d, a 35-kDa physiological fragment of C3b. Size distribution analyses of mixtures of SCR-16/20 or FH with C3d by analytical ultracentrifugation in 50 and 137 mM NaCl buffer revealed a range of discrete peaks, showing that multimeric complexes had formed at physiologically relevant concentrations. Surface plasmon resonance studies showed that native FH binds C3d in two stages. An equilibrium dissociation constant K(D)(1) of 2.6 microM in physiological buffer was determined for the first stage. Overlay experiments indicated that C3d formed multimeric complexes with FH. X-ray scattering showed that the maximum dimension of the C3d complexes with SCR-16/20 at 29 nm was not much longer than that of the unbound SCR-16/20 dimer. Molecular modelling suggested that the ultracentrifugation and scattering data are most simply explained in terms of associating dimers of each of SCR-16/20 and C3d. We conclude that the physiological interaction between FH and C3d is not a simple 1:1 binding stoichiometry between the two proteins that is often assumed. Because the multimers involve the C-terminus of FH, which is bound to host cell surfaces, our results provide new insight on FH regulation during excessive complement activation, both in the fluid phase and at host cell surfaces decorated by C3d.

Hematopoietic growth factor (HGF) mimetics offer a number of attractive advantages as therapeutic agents. Small chemical compounds, in particular, provide reduced cost and oral availability. As many of these mimetics are unrelated in structure to the normal cytokine the immunogenic response is not a significant issue. Isolation of small peptide agonists for erythropoietin (EPO) and thrombopoietin (TPO) receptors has been associated with significant translational challenges and here we summarize approaches used to achieve the potency and stability required for clinical utility. We also compare and contrast the initial screening approaches, and the translational and clinical issues associated with two recently approved TPO mimetics, romiplostim and the orally available eltrombopag. Finally we summarize the development and clinical findings for the EPO mimetic, Hematide, consider alternative approaches, and discuss the future potential for isolation of growth factor (GF) mimetics.

Regulatory T cells (Treg) have recently come to the fore in studies of immune regulation, particularly in autoimmune disease and cancer. While there appear to be several distinct subsets of T cells with regulatory function, a population described as natural Treg and characterized by expression of the transcription factor FOXP3 has attracted particular interest. These cells can be enriched using the surface markers CD4 and CD25, and cord blood is a convenient source of CD25+ Treg. We present detailed protocols for the enrichment of Treg from cord blood using CD25 and a magnetic bead procedure, yielding populations >80% positive for CD25 and 50-65% FOXP3 positive. This enrichment can be followed by a second magnetic bead or a flow sorting step, yielding >95% CD25 and >65% FOXP3 positive populations. Protocols are presented for propagation of these cells in culture (yielding >80% FOXP3 positive cells) and for their phenotypic and functional characterization.

A 5-year-old boy was vaccinated with the Oka strain of varicella zoster virus vaccine before cord blood transplant for chronic granulomatous disease in 2005. In 2006, he developed herpes zoster on his left arm. DNA from the vesicular rash confirmed the Oka vaccine strain of varicella zoster virus caused this complication. He responded well to 10 days of aciclovir treatment.

Borrelia hermsii, the primary etiological agent of tick-borne relapsing fever in North America, binds the complement regulatory protein factor H (FH) as a means of evading opsonophagocytosis and the alternative complement pathway. The ability of FH-binding protein A (FhbA) to bind FH-like protein 1 (FHL-1) has not been assessed previously. In this study, using a whole-cell absorption assay, we demonstrated that B. hermsii absorbs both FH and FHL-1 from human serum. Consistent with this, affinity ligand binding immunoblot analyses revealed that FH constructs spanning short consensus repeats 1 to 7 and 16 to 20 bind to FhbA. To investigate the molecular basis of the interaction of FhbA with FH/FHL-1, recombinant FhbA truncated proteins were generated and tested for FH/FHL-1 binding. Binding required determinants located in both the N- and C-terminal domains of FhbA, suggesting that long-range intramolecular interactions are involved in the formation and presentation of the FH/FHL-1-binding pocket. To identify specific FhbA residues involved in binding, random mutagenesis was performed. These analyses identified a loop region of FhbA that may serve as a contact point for FH/FHL-1. The data presented here expand our understanding of the pathogenic mechanisms of the relapsing fever spirochetes and of the molecular nature of the interaction between FH/FHL-1 and FhbA.

Treponema denticola is an important contributor to periodontal disease. In this study we investigated the ability of T. denticola to bind the complement regulatory proteins factor H and factor H-like protein 1 (FHL-1). The binding of these proteins has been demonstrated to facilitate evasion of the alternative complement cascade and/or to play a role in adherence and invasion. Here we demonstrate that T. denticola specifically binds FHL-1 via a 14-kDa, surface-exposed protein that we designated FhbB. Consistent with its FHL-1 binding specificity, FhbB binds only to factor H recombinant fragments spanning short consensus repeats (SCRs) 1 to 7 (H7 construct) and not to SCR constructs spanning SCRs 8 to 15 and 16 to 20. Binding of H7 to FhbB was inhibited by heparin. The specific involvement of SCR 7 in the interaction was demonstrated using an H7 mutant (H7AB) in which specific charged residues in SCR 7 were replaced by alanine. This construct lost FhbB binding ability. Analyses of the ability of FHL-1 bound to the surface of T. denticola to serve as a cofactor for factor I-mediated cleavage of C3b revealed that C3b is cleaved in an FHL-1/factor I-independent manner, perhaps by an unidentified protease. Based on the data presented here, we hypothesize that the primary function of FHL-1 binding by T. denticola might be to facilitate adherence to FHL-1 present on anchorage-dependent cells and in the extracellular matrix.

The gene encoding the 51-kDa subunit of nicotinamide adenine dinucleotide (NADH) dehydrogenase complex I, a principal component of the mitochondrial electron transport chain, was cloned in Candida tropicalis. The homolog in C. albicans, CaNDH51, was identified, and each allele was successively disrupted by PCR-mediated gene disruption. Wild type, heterozygote, reintegrant, and homozygous null mutants grew as blastoconidia in rich medium containing 3% glucose, but the homozygous null mutant failed to grow in ethanol or acetate. When glucose concentration was varied from 1 mM (0.018%) to 200 mM (3.6%) in a basal salts medium, all strains grew equally well at all glucose concentrations; the wild-type strain, the heterozygote, and the reintegrant exhibited abundant germ tubes, pseudohyphae, and hyphae. In contrast, the ndh51/ndh51 strain failed to display any type of filamentous growth, even in glucose concentrations as low as 1 mM. These results suggest a previously unexplored relationship between mitochondrial electron transport and morphogenesis.

Erythroid tissue is the major site of heme production in the body. The synthesis of heme and globin chains is coordinated at both the transcriptional and post-transcriptional levels to ensure that virtually no free heme or globin protein accumulates. The key rate-controlling enzyme of the heme biosynthetic pathway is 5-aminolevulinate synthase (ALAS) and an erythroid-specific isoform (ALAS2) is up-regulated during erythropoiesis. Differentiation of embryonic stem cells with a disrupted ALAS2 gene has established that expression of this gene is critical for erythropoiesis and cannot be compensated by expression of the ubiquitous isoform of the enzyme (ALAS1). Interestingly, heme appears to be important for expression of globin and other late erythroid genes and for erythroid cell differentiation although the mechanism of this effect is not clear. Transcriptional control elements that regulate the human gene for ALAS2 have been identified both in the promoter and in intronic enhancer regions. Subsequent translation of the ALAS2 mRNA is dependent on an adequate iron supply. The mechanism by which transcription of the gene for ALAS2 is increased by erythropoietin late in erythropoiesis remains an interesting issue. Erythropoietin action may result in altered levels of critical erythroid transcription factors or modulate the phosphorylation/acetylation status of these factors. Defects in the coding region of the gene for ALAS2 underlie the disease state X-linked sideroblastic anemia. In this review, we focus on the regulation and function of erythroid-specific 5-aminolevulinate synthase during erythropoiesis and its role in the X-linked sideroblastic anemia.

Complement factor H (fH) regulates activation of the alternative pathway of C, reducing the amount of C3b deposited on sialic acid-rich surfaces. Heparin binding has been used as a model for examining the sialic acid-binding characteristics of fH. We have previously shown that of the 20 short consensus repeat (SCR) modules of fH, SCR 7 contains an important heparin binding site, but other SCRs also play a role in heparin binding. To localize the other sites, we prepared recombinant truncated and SCR deletion mutants of fH and tested them by heparin-agarose affinity chromatography. The 5 C-terminal SCRs were found to contain a heparin binding site as an SCR 7 deletion mutant of the N terminal 15 SCRs did not bind heparin, but a construct consisting of SCRs 16-20 was shown to bind heparin. Double deletion of SCRs 7 and 20 from fH abrogated binding to heparin, indicating that SCR 20 contains a heparin binding site. This finding was confirmed with the observation that attachment of SCR 20 to a group of nonbinding SCRs produced a heparin-binding protein. A protein consisting of SCRs 19 and 20 did not bind heparin, whereas SCRs 18-20 did, indicating that, although SCR 20 contains a heparin binding site, at least two nonspecific adjacent SCRs are required. fH-related protein-3 (FHR-3) possesses an SCR homologous to SCR 7 of fH and bound heparin, whereas FHR-4, which lacks such an SCR, did not. Thus, fH contains two separate heparin binding sites, which are located in SCRs 7 and 20.

The 5' flanking region of human factor H was cloned using nested polymerase chain reaction (PCR) and the promoter finder method. A total of 1.2 kb has been sequenced and a number of putative regulatory elements identified including glucocorticoid response elements cAMP responsive element, HTF-1, and acute phase signal sequences. A 717 b.p. fragment was cloned into a CAT reporter vector and transfected into HeLa cells. A series of truncations from the 5' end of this fragment were also cloned into the CAT vector. Analysis of CAT activity of the cell lysates showed that the region from -699 to +18 is likely to contain promoter elements for the factor H gene as it was able to drive transcription of the CAT gene.

The present study demonstrates that human astrocytes synthesize and express CD59, a regulator of the membrane attack complex of complement. This was shown by flow cytometry following staining of astrocytes with MAb to CD59, and Western blotting of astrocyte lysates, which revealed the characteristic 18-23,000 M(r) band of CD59. Synthesis of CD59 by astrocytes was confirmed by detection of CD59 specific mRNA by polymerase chain reaction. A low level of C3 deposition occurred on astrocytes following exposure to autologous serum. CD59 may prevent subsequent damage from C5b-9 and protect astrocytes during inflammatory and infectious disorders of the nervous system.

Expression of membrane cofactor protein (CD46) on cultured human astrocytes was demonstrated by indirect immunofluorescence microscopy and flow cytometry following staining with a monoclonal antibody specific for CD46. Western transfer and immunoblotting detected a doublet of Mr 66,000 and 56,000. Analysis of astrocyte mRNA revealed the presence of multiple alternatively spliced transcripts encoding different extracellular regions or cytoplasmic tails of CD46. Astrocytes were also shown to express decay accelerating factor, but not the type 1 complement receptor. Upregulation of astrocyte CD46 occurred following cytomegalovirus infection. These results indicate that astrocytes express proteins involved in regulation of complement activation and protection against autologous complement.