The C1q and gC1qR axis as a novel checkpoint inhibitor in cancer.

Understanding at the molecular level of the cell biology of tumors has led to significant treatment advances in the past. Despite such advances however, development of therapy resistance and tumor recurrence are still unresolved major challenges. This therefore underscores the need to identify novel tumor targets and develop corresponding therapies to supplement existing biologic and cytotoxic approaches so that a deeper and more sustained treatment responses could be achieved. The complement system is emerging as a potential novel target for cancer therapy. Data accumulated to date show that complement proteins, and in particular C1q and its receptors cC1qR/CR and gC1qR/p33/HABP1, are overexpressed in most cancer cells and together are involved not only in shaping the inflammatory tumor microenvironment, but also in the regulation of angiogenesis, metastasis, and cell proliferation. In addition to the soluble form of C1q that is found in plasma, the C1q molecule is also found anchored on the cell membrane of monocytes, macrophages, dendritic cells, and cancer cells, via a 22aa long leader peptide found only in the A-chain. This orientation leaves its 6 globular heads exposed outwardly and thus available for high affinity binding to a wide range of molecular ligands that enhance tumor cell survival, migration, and proliferation. Similarly, the gC1qR molecule is not only overexpressed in most cancer types but is also released into the microenvironment where it has been shown to be associated with cancer cell proliferation and metastasis by activation of the complement and kinin systems. Co-culture of either T cells or cancer cells with purified C1q or anti-gC1qR has been shown to induce an anti-proliferative response. It is therefore postulated that in the tumor microenvironment, the interaction between C1q expressing cancer cells and gC1qR bearing cytotoxic T cells results in T cell suppression in a manner akin to the PD-L1 and PD-1 interaction.

Microbial Protein Binding to gC1qR Drives PLA2G1B-Induced CD4 T-Cell Anergy.

The origin of the impaired CD4 T-cell response and immunodeficiency of HIV-infected patients is still only partially understood. We recently demonstrated that PLA2G1B phospholipase synergizes with the HIV gp41 envelope protein in HIV viremic plasma to induce large abnormal membrane microdomains (aMMDs) that trap and inactivate physiological receptors, such as those for IL-7. However, the mechanism of regulation of PLA2G1B activity by the cofactor gp41 is not known. Here, we developed an assay to directly follow PLA2G1B enzymatic activity on CD4 T-cell membranes. We demonstrated that gp41 directly binds to PLA2G1B and increases PLA2G1B enzymatic activity on CD4 membrane. Furthermore, we show that the conserved 3S sequence of gp41, known to bind to the innate sensor gC1qR, increases PLA2G1B activity in a gC1qR-dependent manner using gC1qR KO cells. The critical role of the 3S motif and gC1qR in the inhibition of CD4 T-cell function by the PLA2G1B/cofactor system in HIV-infected patients led us to screen additional microbial proteins for 3S-like motifs and to study other proteins known to bind to the gC1qR to further investigate the role of the PLA2G1B/cofactor system in other infectious diseases and carcinogenesis. We have thus extended the PLA2G1B/cofactor system to HCV and Staphylococcus aureus infections and additional pathologies where microbial proteins with 3S-like motifs also increase PLA2G1B enzymatic activity. Notably, the bacteria Porphyromonas gingivalis, which is associated with pancreatic ductal adenocarcinoma (PDAC), encodes such a cofactor protein and increased PLA2G1B activity in PDAC patient plasma inhibits the CD4 response to IL-7. Our findings identify PLA2G1B/cofactor system as a CD4 T-cell inhibitor. It involves the gC1qR and disease-specific cofactors which are gC1qR-binding proteins that can contain 3S-like motifs. This mechanism involved in HIV-1 immunodeficiency could play a role in pancreatic cancer and several other diseases. These observations suggest that the PLA2G1B/cofactor system is a general CD4 T-cell inhibitor and pave the way for further studies to better understand the role of CD4 T-cell anergy in infectious diseases and tumor escape.

SARS-CoV-2 Exacerbates COVID-19 Pathology Through Activation of the Complement and Kinin Systems.

Infection with SARS-CoV-2 triggers the simultaneous activation of innate inflammatory pathways including the complement system and the kallikrein-kinin system (KKS) generating in the process potent vasoactive peptides that contribute to severe acute respiratory syndrome (SARS) and multi-organ failure. The genome of SARS-CoV-2 encodes four major structural proteins - the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein. However, the role of these proteins in either binding to or activation of the complement system and/or the KKS is still incompletely understood. In these studies, we used: solid phase ELISA, hemolytic assay and surface plasmon resonance (SPR) techniques to examine if recombinant proteins corresponding to S1, N, M and E: (a) bind to C1q, gC1qR, FXII and high molecular weight kininogen (HK), and (b) activate complement and/or the KKS. Our data show that the viral proteins: (a) bind C1q and activate the classical pathway of complement, (b) bind FXII and HK, and activate the KKS in normal human plasma to generate bradykinin and (c) bind to gC1qR, the receptor for the globular heads of C1q (gC1q) which in turn could serve as a platform for the activation of both the complement system and KKS. Collectively, our data indicate that the SARS-CoV-2 viral particle can independently activate major innate inflammatory pathways for maximal damage and efficiency. Therefore, if efficient therapeutic modalities for the treatment of COVID-19 are to be designed, a strategy that includes blockade of the four major structural proteins may provide the best option.

Low Glucose Mediated Fluconazole Tolerance in Cryptococcus neoformans.

Chronic meningoencephalitis is caused by Cryptococcus neoformans and is treated in many parts of the world with fluconazole (FLC) monotherapy, which is associated with treatment failure and poor outcome. In the host, C. neoformans propagates predominantly under low glucose growth conditions. We investigated whether low glucose, mimicked by growing in synthetic media (SM) with 0.05% glucose (SMlowglu), affects FLC-resistance. A > 4-fold increase in FLC tolerance was observed in seven C. neoformans strains when minimum inhibitory concentration (MIC) was determined in SMlowglu compared to MIC in SM with normal (2%) glucose (SMnlglu). In SMlowglu, C. neoformans cells exhibited upregulation of efflux pump genes AFR1 (8.7-fold) and AFR2 (2.5-fold), as well as decreased accumulation (2.6-fold) of Nile Red, an efflux pump substrate. Elevated intracellular ATP levels (3.2-fold and 3.4-fold), as well as decreased mitochondrial reactive oxygen species levels (12.8-fold and 17-fold), were found in the presence and absence of FLC, indicating that low glucose altered mitochondrial function. Fluorescence microscopy revealed that mitochondria of C. neoformans grown in SMlowglu were fragmented, whereas normal glucose promoted a reticular network of mitochondria. Although mitochondrial membrane potential (MMP) was not markedly affected in SMlowglu, it significantly decreased in the presence of FLC (12.5-fold) in SMnlglu, but remained stable in SMlowglu-growing C. neoformans cells. Our data demonstrate that increased FLC tolerance in low glucose-growing C. neoformans is the result of increased efflux pump activities and altered mitochondrial function, which is more preserved in SMlowglu. This mechanism of resistance is different from FLC heteroresistance, which is associated with aneuploidy of chromosome 1 (Chr1).

Fibers Generated by Plasma Des-AA Fibrin Monomers and Protofibril/Fibrinogen Clusters Bind Platelets: Clinical and Nonclinical Implications.

Objective Soluble fibrin (SF) is a substantial component of plasma fibrinogen (fg), but its composition, functions, and clinical relevance remain unclear. The study aimed to evaluate the molecular composition and procoagulant function(s) of SF. Materials and Methods Cryoprecipitable, SF-rich (FR) and cryosoluble, SF-depleted (FD) fg isolates were prepared and adsorbed on one hydrophilic and two hydrophobic surfaces and scanned by atomic force microscopy (AFM). Standard procedures were used for fibrin polymerization, crosslinking by factor XIII, electrophoresis, and platelet adhesion. Results Relative to FD fg, thrombin-induced polymerization of FR fg was accelerated and that induced by reptilase was markedly delayed, attributable to its decreased (fibrinopeptide A) FpA. FR fg adsorption to each surface yielded polymeric clusters and co-cryoprecipitable solitary monomers. Cluster components were crosslinked by factor XIII and comprised ≤21% of FR fg. In contrast to FD fg, FR fg adsorption on hydrophobic surfaces resulted in fiber generation enabled by both clusters and solitary monomers. This began with numerous short protofibrils, which following prolonged adsorption increased in number and length and culminated in surface-linked three-dimensional fiber networks that bound platelets. Conclusion The abundance of adsorbed protofibrils resulted from (1) protofibril/fg clusters whose fg was dissociated during adsorption, and (2) adsorbed des-AA monomers that attracted solution counterparts initiating protofibril assembly and elongation by their continued incorporation. The substantial presence of both components in transfused plasma and cryoprecipitate augments hemostasis by accelerating thrombin-induced fibrin polymerization and by tightly anchoring the resulting clot to the underlying wound or to other abnormal vascular surfaces.

Loss of Mucosal p32/gC1qR/HABP1 Triggers Energy Deficiency and Impairs Goblet Cell Differentiation in Ulcerative Colitis.

BACKGROUND & AIMS: Cell differentiation in the colonic crypt is driven by a metabolic switch from glycolysis to mitochondrial oxidation. Mitochondrial and goblet cell dysfunction have been attributed to the pathology of ulcerative colitis (UC). We hypothesized that p32/gC1qR/HABP1, which critically maintains oxidative phosphorylation, is involved in goblet cell differentiation and hence in the pathogenesis of UC. METHODS: Ex vivo, goblet cell differentiation in relation to p32 expression and mitochondrial function was studied in tissue biopsies from UC patients versus controls. Functional studies were performed in goblet cell-like HT29-MTX cells in vitro. Mitochondrial respiratory chain complex V-deficient, ATP8 mutant mice were utilized as a confirmatory model. Nutritional intervention studies were performed in C57BL/6 mice. RESULTS: In UC patients in remission, colonic goblet cell differentiation was significantly decreased compared to controls in a p32-dependent manner. Plasma/serum L-lactate and colonic pAMPK level were increased, pointing at high glycolytic activity and energy deficiency. Consistently, p32 silencing in mucus-secreting HT29-MTX cells abolished butyrate-induced differentiation and induced a shift towards glycolysis. In ATP8 mutant mice, colonic p32 expression correlated with loss of differentiated goblet cells, resulting in a thinner mucus layer. Conversely, feeding mice an isocaloric glucose-free, high-protein diet increased mucosal energy supply that promoted colonic p32 level, goblet cell differentiation and mucus production. CONCLUSION: We here describe a new molecular mechanism linking mucosal energy deficiency in UC to impaired, p32-dependent goblet cell differentiation that may be therapeutically prevented by nutritional intervention.

Replicative Aging Remodels the Cell Wall and Is Associated with Increased Intracellular Trafficking in Human Pathogenic Yeasts.

Replicative aging is an underexplored field of research in medical mycology. Cryptococcus neoformans (Cn) and Candida glabrata (Cg) are dreaded fungal pathogens that cause fatal invasive infections. The fungal cell wall is essential for yeast viability and pathogenesis. In this study, we provide data characterizing age-associated modifications to the cell wall of Cn and Cg. Here, we report that old yeast cells upregulate genes of cell wall biosynthesis, leading to cell wall reorganization and increased levels of all major components, including glucan, chitin, and its derivatives, as well as mannan. This results in a significant thickening of the cell wall in aged cells. Old-generation yeast cells exhibited drastic ultrastructural changes, including the presence of abundant vesicle-like particles in the cytoplasm, and enlarged vacuoles with altered pH homeostasis. Our findings suggest that the cell wall modifications could be enabled by augmented intracellular trafficking. This work furthers our understanding of the cell phenotype that emerges during aging. It highlights differences in these two fungal pathogens and elucidates mechanisms that explain the enhanced resistance of old cells to antifungals and phagocytic attacks. IMPORTANCE Cryptococcus neoformans and Candida glabrata are two opportunistic human fungal pathogens that cause life-threatening diseases. During infection, both microorganisms have the ability to persist for long periods, and treatment failure can occur even if standard testing identifies the yeasts to be sensitive to antifungals. Replicative life span is a trait that is measured by the number of divisions a cell undergoes before death. Aging in fungi is associated with enhanced tolerance to antifungals and resistance to phagocytosis, and characterization of old cells may help identify novel antifungal targets. The cell wall remains an attractive target for new therapies because it is essential for fungi and is not present in humans. This study shows that the organization of the fungal cell wall changes remarkably during aging and becomes thicker and is associated with increased intracellular trafficking as well as the alteration of vacuole morphology and pH homeostasis.

Anti gC1qR/p32/HABP1 Antibody Therapy Decreases Tumor Growth in an Orthotopic Murine Xenotransplant Model of Triple Negative Breast Cancer.

gC1qR is highly expressed in breast cancer and plays a role in cancer cell proliferation. This study explored therapy with gC1qR monoclonal antibody 60.11, directed against the C1q binding domain of gC1qR, in a murine orthotopic xenotransplant model of triple negative breast cancer. MDA231 breast cancer cells were injected into the mammary fat pad of athymic nu/nu female mice. Mice were segregated into three groups (n = 5, each) and treated with the vehicle (group 1) or gC1qR antibody 60.11 (100 mg/kg) twice weekly, starting at day 3 post-implantation (group 2) or when the tumor volume reached 100 mm3 (group 3). At study termination (d = 35), the average tumor volume in the control group measured 895 ± 143 mm3, compared to 401 ± 48 mm3 and 701 ± 100 mm3 in groups 2 and 3, respectively (p < 0.05). Immunohistochemical staining of excised tumors revealed increased apoptosis (caspase 3 and TUNEL staining) in 60.11-treated mice compared to controls, and decreased angiogenesis (CD31 staining). Slightly decreased white blood cell counts were noted in 60.11-treated mice. Otherwise, no overt toxicities were observed. These data are the first to demonstrate an in vivo anti-tumor effect of 60.11 therapy in a mouse model of triple negative breast cancer.

The C1q Receptors: Focus on gC1qR/p33 (C1qBP, p32, HABP-1)1.

In the past several years, a number of C1q binding surface proteins or receptors have been described. This is not of course surprising considering the complexity of the C1q molecule and its ability to bind to a wide range of cellular and plasma proteins via both its collagen-like [cC1q] region and its heterotrimeric globular heads [gC1q] each of which in turn is capable of binding a specific ligand. However, while each of these "receptor" molecules undoubtedly plays a specific function within its restricted microenvironment, and therefore merits full attention, this review nonetheless, will singularly focus on the structure and function of gC1qR-a multi-functional and multi-compartmental protein, which plays an important role in inflammation, infection, and cancer. Although first identified as a receptor for C1q, gC1qR has been shown to bind to a plethora of proteins found in plasma, on the cell surface and on pathogenic microorganisms. The plasma proteins that bind to gC1qR are mostly blood coagulation proteins and include high molecular weight kininogen [HK], Factor XII [Hageman factor], fibrinogen, thrombin [FII], and multimeric vitronectin. This suggests that gC1qR can play an important role in modulating not only of fibrin formation, particularly at local sites of immune injury and/or inflammation, but by activating the kinin/kallikrein system, it is also able to generate, bradykinin, a powerful vasoactive peptide that is largely responsible for the swelling seen in angioedema. Another important function of gC1qR is in cancer, where it has been shown to play a role in tumor cell survival, growth and metastatic invasion by interacting with critical molecules in the tumor cell microenvironment including those of the complement system and kinin system. Finally, by virtue of its ability to interact with a growing list of pathogen-associated molecules, including bacterial and viral ligands, gC1qR is becoming recognized as an important pathogen recognition receptor [PRR]. Given the numerous roles it plays in a growing list of disease settings, gC1qR has now become a potential target for the development of monoclonal antibody-based and/or small molecule-based therapies.

New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in 3D cancer spheroids induced by purified colon tumor-initiating cells.

BACKGROUND: Growing evidence suggests that the majority of tumors are organized hierarchically, comprising a population of tumor-initiating, or cancer stem cells (CSCs) responsible for tumor development, maintenance and resistance to drugs. Previously we have shown that the CD133high/CD44high fraction of colon cancer cells is different from their bulk counterparts at the functional, morphological and genomic levels. In contrast to the majority of colon cancer cells expressing moderate levels of CD133, CD44 and CD166, cells with a high combined expression of CD133 and CD44 possessed several characteristic stem cell features, including profound self-renewal capacity in vivo and in vitro, and the ability to give rise to different cell phenotypes. The present study was undertaken for two aims: a) to determine stem cell-related genomic characteristics of floating 3D multicellular spheroids induced by CD133high/CD44high colon cancer cells; and b) to evaluate CSC-specific alterations induced by new-generation taxoid SB-T-1214. RESULTS: Selected CSC phenotype was isolated from three independent invasive colon cancer cell lines, HCT116, HT29 and DLD-1. A stem cell-specific PCR array assay (SABiosciences) revealed that colonospheres induced by purified CD133high/CD44high expressing cells display profound up-regulation of stem cell-related genes in comparison with their bulk counterparts. The FACS analysis has shown that the 3D colonospheres contained some minority cell populations with high levels of expression of Oct4, Sox2, Nanog and c-Myc, which are essential for stem cell pluripotency and self-renewal. Single administration of the SB-T-1214 at concentration 100 nM-1 microM for 48 hr not only induced growth inhibition and apoptotic cell death in these three types of colon cancer spheroids in 3D culture, but also mediated massive inhibition of the stem cell-related genes and significant down-regulation of the pluripotency gene expression. PCR array and FACS data were confirmed with western blotting. Importantly, viable cells that survived this treatment regimen were no longer able to induce secondary floating spheroids and exhibited significant morphological abnormalities. CONCLUSIONS: We report here that a new-generation taxoid SB-T-1214 possesses significant activity against colon cancer spheroids induced by and enriched with drug resistant tumorigenic CD133high/CD44high cells and efficiently inhibited expression of the majority of stem cell-related genes. Our data indicates that the previously observed long-term efficacy of SB-T-1214 against drug resistant colon tumors in vivo may be explained by the down-regulation of multiple stem cell-related genes in the tumorigenic cell population, in addition to its known efficacy as a mitotic poison against proliferating cancer cells.

Francisella tularensis infection-derived monoclonal antibodies provide detection, protection, and therapy.

Francisella tularensis is the causative agent of tularemia and a potential agent of biowarfare. As an easily transmissible infectious agent, rapid detection and treatment are necessary to provide a positive clinical outcome. As an agent of biowarfare, there is an additional need to prevent infection. We made monoclonal antibodies to the F. tularensis subsp. holarctica live vaccine strain (F. tularensis LVS) by infecting mice with a sublethal dose of bacteria and, following recovery, by boosting the mice with sonicated organisms. The response to the initial and primary infection was restricted to immunoglobulin M antibody directed solely against lipopolysaccharide (LPS). After boosting with sonicated organisms, the specificity repertoire broadened against protein antigens, including DnaK, LpnA, FopA, bacterioferritin, the 50S ribosomal protein L7/L12, and metabolic enzymes. These monoclonal antibodies detect F. tularensis LVS by routine immunoassays, including enzyme-linked immunosorbent assay, Western blot analysis, and immunofluorescence. The ability of the antibodies to protect mice from intradermal infection, both prophylactically and therapeutically, was examined. An antibody to LPS which provides complete protection from infection with F. tularensis LVS and partial protection from infection with F. tularensis subsp. tularensis strain SchuS4 was identified. There was no bacteremia and reduced organ burden within the first 24 h when mice were protected from F. tularensis LVS infection with the anti-LPS antibody. No antibody that provided complete protection when administered therapeutically was identified; however, passive transfer of antibodies against LPS, FopA, and LpnA resulted in 40 to 50% survival of mice infected with F. tularensis LVS.

Francisella tularensis has a significant extracellular phase in infected mice.

The ability of Francisella tularensis to replicate in macrophages has led many investigators to assume that it resides primarily intracellularly in the blood of mammalian hosts. We have found this supposition to be untrue. In almost all cases, the majority of F. tularensis recovered from the blood of infected mice was in plasma rather than leukocytes. This distribution was observed irrespective of size of inoculum, route of inoculation, time after inoculation, or virulence of the infecting strain. Our findings yield new insight into the pathogenesis of tularemia and may have important ramifications in the search for anti-Francisella therapies.

Gene expression profiling in conjunction with physiological rescues of IKKalpha-null cells with wild type or mutant IKKalpha reveals distinct classes of IKKalpha/NF-kappaB-dependent genes.

Cellular responses to stress-like stimuli require the IkappaB kinase (IKK) signalsome (IKKalpha, IKKbeta, and NEMO/IKKgamma) to activate NF-kappaB-dependent genes. IKKbeta and NEMO/IKKgamma are required to release NF-kappaB p65/p50 heterodimers from IkappaBalpha, resulting in their nuclear migration and sequence-specific DNA binding; but IKKalpha was found to be dispensable for this initial phase of canonical NF-kappaB activation. Nevertheless, IKKalpha-/- mouse embryonic fibroblasts (MEFs) fail to express NF-kappaB targets in response to proinflammatory stimuli, uncovering a nuclear role for IKKalpha in NF-kappaB activation. However, it remains unknown whether the global defect in NF-kappaB-dependent gene expression of IKKalpha-/- cells is caused by the absence of IKKalpha kinase activity. We show by gene expression profiling that rescue of near physiological levels of wild type IKKalpha in IKKalpha-/- MEFs globally restores expression of their canonical NF-kappaB target genes. To prove that the kinase activity of IKKalpha was required on a genomic scale, the same physiological rescue was performed with a kinase-dead, ATP binding domain IKKalpha mutant (IKKalpha(K44M)). Remarkably, the IKKalpha(K44M) protein rescued approximately 28% of these genes, albeit in a largely stimulus-independent manner with the notable exception of several genes that also acquired tumor necrosis factor-alpha responsiveness. Thus the IKKalpha-containing signalsome unexpectedly functions in the presence and absence of extracellular signals in both kinase-dependent and -independent modes to differentially modulate the expression of five distinct classes of IKKalpha/NF-kappaB-dependent genes.

LcrV capture enzyme-linked immunosorbent assay for detection of Yersinia pestis from human samples.

In the United States, there is currently a major gap in the diagnostic capabilities with regard to plague. To address this, we developed an antigen capture assay using an essential virulence factor secreted by Yersinia spp., LcrV, as the target antigen. We generated anti-LcrV monoclonal antibodies (MAbs) and screened them for the ability to bind bacterially secreted native Yersinia pestis LcrV. Anti-LcrV MAb 19.31 was used as a capture antibody, and biotinylated MAb 40.1 was used for detection. The detection limit of this highly sensitive Yersinia LcrV capture enzyme-linked immunosorbent assay is 0.1 ng/ml. The assay detected LcrV from human sputum and blood samples treated with concentrations as low as 0.5 ng/ml of bacterially secreted native Y. pestis LcrV. This assay could be used as a tool to help confirm the diagnosis of plague in patients presenting with pneumonia.

Extrusion of an imperfect palindrome to a cruciform in superhelical DNA: complete determination of energetics using a statistical mechanical model.

We present a detailed study of the extrusion of an imperfect palindrome, derived from the terminal regions of vaccinia virus DNA and contained in a superhelical plasmid, into a cruciform containing bulged bases. We monitor the course of extrusion by two-dimensional gel electrophoresis experiments as a function of temperature and linking number. We find that extrusion pauses at partially extruded states as negative superhelicity increases. To understand the course of extrusion with changes in linking number, DeltaLk, we present a rigorous semiempirical statistical mechanical analysis that includes complete coupling between DeltaLk, cruciform extrusion, formation of extrahelical bases, and temperature-dependent denaturation. The imperfections in the palindrome are sequentially incorporated into the cruciform arms as hairpin loops, single unpaired bases, and complex local regions containing several unpaired bases. We analyze the results to determine the free energies, enthalpies and entropies of formation of all local structures involved in extrusion. We find that, for each unpaired structure, the DeltaG, DeltaH and DeltaS of formation are all approximately proportional to the number of unpaired bases contained therein. This surprising result holds regardless of the arrangement or composition of unpaired bases within a particular structure. Imperfections have major effects on the overall energetics of cruciform extrusion and on the course of this transition. In particular, the extent of the DeltaLk change necessary to extrude an imperfect palindrome is considerably greater than that required for extrusion of the underlying perfect palindrome. Our analysis also suggests that, at higher temperatures, significant denaturation at the base of an imperfect cruciform can successfully compete with extension of the cruciform arms.