Blocking TNF signaling may save lives in COVID-19 infection.

Global vaccination effort and better understanding of treatment strategies provided a ray of hope for improvement in COVID-19 pandemic, however, in many countries, the disease continues to collect its death toll. The major pathogenic mechanism behind severe cases associated with high mortality is the burst of pro-inflammatory cytokines TNF, IL-6, IFNγ and others, resulting in multiple organ failure. Although the exact contribution of each cytokine is not clear, we provide an evidence that the central mediator of cytokine storm and its devastating consequences may be TNF. This cytokine is known to be involved in activated blood clotting, lung damage, insulin resistance, heart failure, and other conditions. A number of currently available pharmaceutical agents such as monoclonal antibodies and soluble TNF receptors can effectively prevent TNF from binding to its receptor(s). Other drugs are known to block NFkB, the major signal transducer molecule used in TNF signaling, or to block kinases involved in downstream activation cascades. Some of these medicines have already been selected for clinical trials, but more work is needed. A simple, rapid, and inexpensive method of directly monitoring TNF levels may be a valuable tool for a timely selection of COVID-19 patients for anti-TNF therapy.

Detection of ß cell death in diabetes using differentially methylated circulating DNA.

In diabetes mellitus, ß cell destruction is largely silent and can be detected only after significant loss of insulin secretion capacity. We have developed a method for detecting ß cell death in vivo by amplifying and measuring the proportion of insulin 1 DNA from ß cells in the serum. By using primers that are specific for DNA methylation patterns in ß cells, we have detected circulating copies of ß cell-derived demethylated DNA in serum of mice by quantitative PCR. Accordingly, we have identified a relative increase of ß cell-derived DNA after induction of diabetes with streptozotocin and during development of diabetes in nonobese diabetic mice. We have extended the use of this assay to measure ß cell-derived insulin DNA in human tissues and serum. We found increased levels of demethylated insulin DNA in subjects with new-onset type 1 diabetes compared with age-matched control subjects. Our method provides a noninvasive approach for detecting ß cell death in vivo that may be used to track the progression of diabetes and guide its treatment.

NKG2A is a marker for acquisition of regulatory function by human CD8+ T cells activated with anti-CD3 antibody.

Treatment with anti-CD3 mAb modulates immune responses that cause type 1 diabetes and other diseases. CD8+ Tregs can be induced in vitro and in vivo by mAb. However, 1/3 of patients do not respond to drug therapy and in an equal proportion, anti-CD3 mAb does not induce Tregs in vitro. The acquisition of CD8+ Treg activity is a function of the CD8+ cells and not the targets in the assay. To identify markers to differentiate responses of CD8+ Tregs, we analyzed genes differentially expressed in CD8+ T cells of non-responders compared with responders, and found that an inhibitory receptor NKG2A (CD159a) was highly expressed in cells from all non-responders tested. Application of a mAb agonistic to NKG2A during in vitro CD8+ Treg induction by anti-CD3 prevented induction of CD8+ Tregs. CD8+ T cells that are TNFR2+ but NKG2A- are the most potently induced Tregs. The level of NKG2A expression on resting CD8+ T cells inversely correlated with acquisition of regulatory function when activated. We suggest that the induction of human CD8+ Tregs by anti-CD3 mAb is controlled by a negative signaling through NKG2A, and that NKG2A may serve as a negative marker of human CD8+ Tregs.

Acquisition of regulatory function by human CD8(+) T cells treated with anti-CD3 antibody requires TNF.

Anti-CD3 mAb can modulate graft rejection and attenuate autoimmune diseases but their mechanism(s) of action remain unclear. CD8(+) T cells with regulatory function are induced in vitro by Teplizumab, a humanized anti-CD3 antibody and inhibit responses of autologous and allogeneic T cells. They inhibit CD4(+) T-cell proliferation by mechanisms involving TNF and CCL4, and by blocking target cell entry into G2/M phase of cell cycle but neither kill them, nor compete for IL-2. CD8(+) Treg can be isolated from peripheral blood following treatment of patients with Type 1 diabetes with Teplizumab, but not from untreated patients. The induction of CD8(+) Treg by anti-CD3 mAb requires TNF and signaling through the NF-κB cascade. The CD8(+) Treg express CD25, glucocorticoid-induced TNF receptor family, CTLA-4, Foxp3, and TNFR2, and the combined expression of TNFR2 and CD25 identifies a potent subpopulation of CD8(+) Treg. These studies have identified a novel mechanism of immune regulation by anti-CD3 mAb and markers that may be used to track inducible CD8(+) Treg in settings such as chronic inflammation or immune therapy.

A competitive ligand-binding assay to detect neutralizing antibodies to a bispecific drug using a multiplex Meso Scale Discovery platform.

Background: Monitoring appearance of neutralizing antibodies (NAbs) to multidomain large molecule drugs is a challenging task. Materials & methods: Here, we report development of a competitive ligand-binding assay for detection of NAbs to a bispecific candidate drug using a multiplex Meso Scale Discovery platform, which allows for detection of NAbs to both drug arms in the same sample. Results: The assay has sensitivity better than 250 ng/ml and is tolerant to the presence of drug at concentration >600 µg/ml and to the level of soluble target(s) >400 ng/ml. Conclusion: Our data suggest that multiplex approach can be successfully used for development of NAb assays in competitive ligand-binding assay format.

Generation and function of human regulatory CD8+ T cells induced by a humanized OKT3 monoclonal antibody hOKT3gamma1 (Ala-Ala).

Recent studies with a humanized anti-CD3 antibody in clinical trials suggested that this drug can induce regulatory CD8+ T cells. In this review, we discuss how these regulatory human CD8+ cells can be generated and the possible molecular tools they may use to inhibit immune responses. We compare our data on CD8+ regulatory cells induced by anti-CD3 stimulation with those reported in other experimental systems to identify both common and unique features of these cells.

Autoimmunity and beta cell regeneration in mouse and human type 1 diabetes: the peace is not enough.

Accumulating data from animal models of type 1 diabetes and some findings from clinical studies suggest that autoimmune destruction of islet beta cells is associated with enhanced beta cell regeneration. Successful immune therapies, aimed at preservation of islet cell mass, result in a remarkable reduction of beta cell regeneration. Treated or not, as long as the task of treatment is limited by "making peace" with autoimmunity, the process of beta cell loss continues. Additional therapeutic modalities capable of stimulating beta cell regeneration in the absence of active autoimmune destruction are urgently needed.

Acute rejection of white adipose tissue allograft.

White adipose tissue (WAT) transplantation, although widely used in humans, has been done for cosmetic and reconstructive purposes only. Accumulating evidence indicates, however, that WAT is an important endocrine organ and, therefore, WAT transplantation may become valuable as a replacement therapy for a number of hereditary human diseases. Because the most readily available source for such transplantations would be allogeneic tissue, the mechanisms involved in the rejection of WAT allograft should be explored. We have established a model in which leptin-producing allogeneic WAT is transplanted into leptin-deficient ob/ob mice. Because ob/ob mice are obese, hyperphagic, and hypothermic, WAT allograft function is monitored as the reversal of this leptin-deficient phenotype. Here we report that allografted WAT is primarily nonfunctional. However, when WAT is transplanted into immunodeficient (Rag1-/-) ob/ob mice, or into ob/ob mice depleted of T cells by anti-CD3 antibody, a long-term graft survival is achieved as indicated by the reversal of hyperphagia, weight loss, and normalization of body temperature. The symptoms of leptin deficiency rapidly recur when normal spleen cells of the recipient type are injected, or when the antibody treatment is terminated. In contrast, selective depletion of either CD4+ or CD8+ cells alone does not prevent WAT allograft rejection. Similarly, WAT allografts that do not express MHC class I or class II molecules are rapidly rejected, suggesting that both CD4+ and CD8+ T cells may independently mediate WAT allograft rejection.

Yersinia pestis Caf1 Protein: Effect of Sequence Polymorphism on Intrinsic Disorder Propensity, Serological Cross-Reactivity and Cross-Protectivity of Isoforms.

Yersinia pestis Caf1 is a multifunctional protein responsible for antiphagocytic activity and is a key protective antigen. It is generally conserved between globally distributed Y. pestis strains, but Y. pestis subsp. microtus biovar caucasica strains circulating within populations of common voles in Georgia and Armenia were reported to carry a single substitution of alanine to serine. We investigated polymorphism of the Caf1 sequences among other Y. pestis subsp. microtus strains, which have a limited virulence in guinea pigs and in humans. Sequencing of caf1 genes from 119 Y. pestis strains belonging to different biovars within subsp. microtus showed that the Caf1 proteins exist in three isoforms, the global type Caf1NT1 (Ala48 Phe117), type Caf1NT2 (Ser48 Phe117) found in Transcaucasian-highland and Pre-Araks natural plague foci #4-7, and a novel Caf1NT3 type (Ala48 Val117) endemic in Dagestan-highland natural plague focus #39. Both minor types are the progenies of the global isoform. In this report, Caf1 polymorphism was analyzed by comparing predicted intrinsic disorder propensities and potential protein-protein interactivities of the three Caf1 isoforms. The analysis revealed that these properties of Caf1 protein are minimally affected by its polymorphism. All protein isoforms could be equally detected by an immunochromatography test for plague at the lowest protein concentration tested (1.0 ng/mL), which is the detection limit. When compared to the classic Caf1NT1 isoform, the endemic Caf1NT2 or Caf1NT3 had lower immunoreactivity in ELISA and lower indices of self- and cross-protection. Despite a visible reduction in cross-protection between all Caf1 isoforms, our data suggest that polymorphism in the caf1 gene may not allow the carriers of Caf1NT2 or Caf1NT3 variants escaping from the Caf1NT1-mediated immunity to plague in the case of a low-dose flea-borne infection.

Two Isoforms of Yersinia pestis Plasminogen Activator Pla: Intraspecies Distribution, Intrinsic Disorder Propensity, and Contribution to Virulence.

It has been shown previously that several endemic Y. pestis isolates with limited virulence contained the I259 isoform of the outer membrane protease Pla, while the epidemic highly virulent strains possessed only the T259 Pla isoform. Our sequence analysis of the pla gene from 118 Y. pestis subsp. microtus strains revealed that the I259 isoform was present exclusively in the endemic strains providing a convictive evidence of more ancestral origin of this isoform. Analysis of the effects of the I259T polymorphism on the intrinsic disorder propensity of Pla revealed that the I259T mutation slightly increases the intrinsic disorder propensity of the C-terminal tail of Pla and makes this protein slightly more prone for disorder-based protein-protein interactions, suggesting that the T259 Pla could be functionally more active than the I259 Pla. This assumption was proven experimentally by assessing the coagulase and fibrinolytic activities of the two Pla isoforms in human plasma, as well as in a direct fluorometric assay with the Pla peptide substrate. The virulence testing of Pla-negative or expressing the I259 and T259 Pla isoforms Y. pestis subsp. microtus and subsp. pestis strains did not reveal any significant difference in LD50 values and dose-dependent survival assays between them by using a subcutaneous route of challenge of mice and guinea pigs or intradermal challenge of mice. However, a significant decrease in time-to-death was observed in animals infected with the epidemic T259 Pla-producing strains as compared to the parent Pla-negative variants. Survival curves of the endemic I259 Pla+ strains fit between them, but significant difference in mean time to death post infection between the Pla-strains and their I259 Pla+ variants could be seen only in the isogenic set of subsp. pestis strains. These findings suggest an essential role for the outer membrane protease Pla evolution in Y. pestis bubonic infection exacerbation that is necessary for intensification of epidemic process from endemic natural focality with sporadic cases in men to rapidly expanding epizootics followed by human epidemic outbreaks, local epidemics or even pandemics.

Adipose tissue transplantation protects ob/ob mice from obesity, normalizes insulin sensitivity and restores fertility.

Adipose tissue affects metabolism by secreting various adipokines. Lipodystropic mice benefit both from leptin replacement therapy and from transplantation of normal fat. Leptin-deficient Lep(ob)/Lep(ob) (ob/ob) mice can also be treated with leptin. Surprisingly, there have been no reports of successful treatment of obese ob/ob mice by transplantation of normal white adipose tissue (WAT). If WAT transplantation is ineffective in treating insulin resistance and diabetes in obese individuals, its applicability may be limited in humans as such abnormalities are usually associated with obesity. In the current study, we tested whether WAT transplantation might prevent, and even reverse, abnormalities characteristic of ob/ob mice. To assess the preventive potential, 6-week-old ob/ob mice were transplanted, subcutaneously, with gonadal fat pads from normal mice. Profound effects on multiple physiological phenotypes were achieved despite leptin levels below 25% of those in control mice. WAT from one donor reduced body weight gain, and WAT from 4 or 8 donors prevented obesity in ob/ob mice. Nonfasting insulin levels and insulin tolerance test were normalized. Corticosterone elevation was also prevented. Finally, WAT from 4 donors restored fertility in ob/ob females. The effects of WAT transplantation were long-lasting, with body weight gain suppressed for at least 40 weeks. To assess the therapeutic potential, obese 13-month-old ob/ob mice with a long history of leptin deficiency were used. Their body weight decreased by approximately 50% when transplanted with WAT from 8 donors. As in young recipients, transplantation greatly reduced nonfasting insulin, suggesting normalized insulin sensitivity. Thus, WAT transplantation was effective for both prevention and therapy. In the future, WAT transplantation may become a useful alternative to hormone replacement in treating not only lipodystropy, but also certain types of obesity.

Functional human to mouse adipose tissue xenotransplantation.

White adipose tissue (WAT) produces a number of metabolically important factors and, therefore, some inborn errors of metabolism may potentially be corrected by transplantation of normal allogeneic WAT. To explore the ability of human WAT (HuWAT) to compensate for a missing factor and to induce allogeneic immune response, we created leptin-deficient, immunodeficient mice and transplanted them with either 2·5 or 5 ml HuWAT. Recipient mice showed stable levels of human leptin in circulation, reduced body mass gain, and amelioration of hepatic steatosis. Food consumption and plasma insulin levels were reduced only in recipients of 5 ml WAT. Transfer of 2×10(7) human mononuclear cells to reject WAT as an allograft was ineffective and resulted only in some reduction of circulating leptin and a limited damage to the WAT grafts followed by the loss of human leukocytes.

Synergistic reversal of type 1 diabetes in NOD mice with anti-CD3 and interleukin-1 blockade: evidence of improved immune regulation.

Inflammatory cytokines are involved in autoimmune diabetes: among the most prominent is interleukin (IL)-1ß. We postulated that blockade of IL-1ß would modulate the effects of anti-CD3 monoclonal antibody (mAb) in treating diabetes in NOD mice. To test this, we treated hyperglycemic NOD mice with F(ab')(2) fragments of anti-CD3 mAb with or without IL-1 receptor antagonist (IL-1RA), or anti-IL-1ß mAb. We studied the reversal of diabetes and effects of treatment on the immune system. Mice that received a combination of anti-CD3 mAb with IL-1RA showed a more rapid rate of remission of diabetes than mice treated with anti-CD3 mAb or IL-1RA alone. Combination-treated mice had increased IL-5, IL-4, and interferon (IFN)-γ levels in circulation. There were reduced pathogenic NOD-relevant V7 peptide-V7(+) T cells in the pancreatic lymph nodes. Their splenocytes secreted more IL-10, had increased arginase expression in macrophages and dendritic cells, and had delayed adoptive transfer of diabetes. After 1 month, there were increased concentrations of IgG1 isotype antibodies and reduced intrapancreatic expression of IFN-γ, IL-6, and IL-17 despite normal splenocyte cytokine secretion. These studies indicate that the combination of anti-CD3 mAb with IL-1RA is synergistic in reversal of diabetes through a combination of mechanisms. The combination causes persistent remission from islet inflammation.

Comparison of human fetal liver, umbilical cord blood, and adult blood hematopoietic stem cell engraftment in NOD-scid/gammac-/-, Balb/c-Rag1-/-gammac-/-, and C.B-17-scid/bg immunodeficient mice.

Immunodeficient mice bearing components of a human immune system present a novel approach for studying human immune responses. We investigated the number, phenotype, developmental kinetics, and function of developing human immune cells following transfer of CD34(+) hematopoietic stem cell (HSC) preparations originating from second trimester human fetal liver (HFL), umbilical cord blood (UCB), or granulocyte colony-stimulating factor-mobilized adult blood (G-CSF-AB) delivered via intrahepatic injection into sublethally irradiated neonatal NOD-scid/gammac(-/-), Balb/c-Rag1(-/-)gammac(-/-), and C.B-17-scid/bg mice. HFL and UCB HSC provided the greatest number and breadth of developing cells. NOD-scid/gammac(-/-) and Balb/c-Rag1(-/-)gammac(-/-) harbored human B and dendritic cells as well as human platelets in peripheral blood, whereas NOD-scid/gammac(-/-) mice harbored higher levels of human T cells. NOD-scid/gammac(-/-) mice engrafted with HFL CD34(+) HSC demonstrated human immunological competence evidenced by white pulp expansion and increases in total human immunoglobulin following immunization with T-dependent antigens and delayed-type hypersensitivity-infiltrating leukocytes in response to antigenic challenge. In conclusion, we describe an encouraging base system for studying human hematopoietic lineage development and function utilizing human HFL or UCB HSC-engrafted NOD-scid/gammac(-/-) mice that is well suited for future studies toward the development of a fully competent humanized mouse model.

Human regulatory CD8 T cells.

Administration of a humanized monoclonal anti-CD3 antibody (mAb) to patients with type 1 diabetes (T1D) increases their C-peptide responses and the CD8/CD4 ratio. Incubation of human peripheral blood mononuclear cells (PBMC) with mAb in vitro has been shown to induce CD8(+) regulatory T cells (Tregs) capable of inhibiting proliferation of CD4(+) T cells. We hypothesized that CD8(+) Tregs function through secretion of cytokines. To test that possibility, we generated CD8(+) Tregs, sorted them by FACS, incubated them with syngeneic CD8-depleted PBMC in the presence of staphylococcal enterotoxin B (SEB), and measured proliferation of T cells and cytokines. Using neutralizing anti-cytokine mAbs, we show that the inhibitory effect of CD8(+) Tregs could be partially alleviated by anti-CCL-4, anti-TNF, and to a lesser extent anti-IL2, suggesting that these cytokines contribute to CD8(+) Treg function.

Reduced adiposity in ob/ob mice following total body irradiation and bone marrow transplantation.

OBJECTIVE: The objective of this study was to assess long-term metabolic consequences of total body irradiation (TBI) and bone marrow transplantation. Severe obesity develops due to both hypertrophy and hyperplasia of adipocytes. We hypothesized that TBI would arrest adipose tissue growth and would affect insulin resistance (IR). RESEARCH METHODS AND PROCEDURES: We exposed 2-month-old female ob/ob mice to 8 Grays of TBI followed by bone marrow transplantation and tested the animals for body weight (BW) gain, body composition, blood glucose, and insulin sensitivity. RESULTS: Two months after TBI, irradiated mice stopped gaining BW, whereas non-treated mice continued to grow. At the age of 9.5 months, body mass of irradiated mice was 60.6 +/- 1.4 grams, which was only 61% of that in non-treated ob/ob controls (99.4 +/- 1.6 grams). Body composition measurements by DXA showed that decreased BW was primarily due to an impaired fat accumulation. This could not result from the production of leptin by bone marrow-derived adipocyte progenitors because inhibition of the obese phenotype was identical in recipients of both B6 and ob/ob bone marrow. Inability of the irradiated mice to accumulate fat was associated with hepatomegaly, lower levels of monocyte chemoattractant protein-1 expression in adipose tissue, and increased IR. DISCUSSION: Our data argue in favor of the hypothesis that inability of adipose tissue to expand may increase IR. This mouse model may be valuable for studies of late-onset radiation-induced IR in humans.

A mouse model of conditional lipodystrophy.

Lipodystrophies are syndromes of adipose tissue degeneration associated with severe defects in lipid and glucose homeostasis. We report here the generation and analysis of Pparg(ldi), a targeted allele that confers conditional dominant lipodystrophy in mice. The Pparg(ldi) allele was generated by insertion of the Tet activator (tTA) and a tTA-regulated Flag-Pparg1 transgene into the Pparg gene. Unexpectedly, tTA elicits mild lipodystrophy, insulin resistance, and dyslipidemia, and the Flag-PPARgamma1 transgene surprisingly exacerbates these traits. Doxycycline can both completely prevent and reverse these phenotypes, providing a mouse model of inducible lipodystrophy. Embryonic fibroblasts from either Pparg(ldi/+) or the phenotypically similar aP2-nSrebp1c (Sr) transgenic mice undergo robust adipogenesis, suggesting that neither strain develops lipodystrophy because of defective adipocyte differentiation. In addition, Pparg(ldi/+) adipose tissue shares extensive gene expression aberrations with that of Sr mice, authenticating the phenotype at the molecular level and revealing a common expression signature of lipodystrophic fat. Thus, the Pparg(ldi/+) mouse provides a conditional animal model for studying lipodystrophy and its associated physiology and gene expression.

Susceptibility to induced and spontaneous carcinogenesis is increased in fatless A-ZIP/F-1 but not in obese ob/ob mice.

Obesity is typically associated with increased tumor susceptibility, whereas caloric restriction, a regimen resulting in leanness, inhibits carcinogenesis. The link between adiposity and malignancies suggests that adipose tissue may influence carcinogenesis. An adipose tissue hormone, leptin, could be procarcinogenic because it stimulates proliferation in various tissues and tumor cell lines. Leptin may contribute to the correlation between adiposity and malignancies as its levels are usually increased in obese subjects and reduced by caloric restriction. We hypothesized that leptin deficiency, despite obesity, would inhibit carcinogenesis in leptin-null ob/ob mice and tested this hypothesis in two models: (a) two-stage skin carcinogenesis initiated by 7,12-dimethylbenz(a)anthracene and promoted by phorbol 12-myristate 13-acetate (PMA) and (b) p53 deficiency. Contrary to a typical association between obesity and enhanced carcinogenesis, obese ob/ob mice developed induced skin papillomas and spontaneous p53-deficient malignancies, mostly lymphomas, similarly to their lean littermates. Surprisingly, lipodystrophic (ZIP) mice that had very little both adipose tissue and leptin were highly susceptible to carcinogenesis. Hyperphagia, hyperinsulinemia, and hyperglycemia are unlikely to have contributed significantly to the enhancement of carcinogenesis in ZIP mice because similarly hyperphagic, hyperinsulinemic, and hyperglycemic ob/ob mice had normal susceptibility to carcinogenesis. Our data suggest that, in contrast to a well-known correlation between obesity and cancer, the direct effect of adipose tissue may rather be protective.

The importance of APC.

Readers in immunology are familiar with the importance of antigen presenting cells in mounting immune responses. For the purpose of this particular editorial article, however, the abbreviation APC will stand for article processing charges. The publisher will introduce APCs for this Journal in May, 2005. Here we explain why article-processing charges are important to maintain our Open Access journal.

Cdc2/cyclin B1 interacts with and modulates inositol 1,4,5-trisphosphate receptor (type 1) functions.

The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the importance of IP3-gated calcium (Ca2+) release to cellular apoptosis. However, the specific upstream biochemical events leading to IP3-gated Ca2+ release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase 1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we show that: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation at Thr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2+ release. Taken together, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2+ signaling. In addition, identification of a CyB docking site(s) on IP3R1 demonstrates, for the first time, a direct interaction between a cell cycle component and an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may constitute a new therapy for the treatment of several human immune disorders.