Wednesday, September 28, 2022

Antibody specificities are Gut reactions

Germ-free (GF) mice have undeveloped immune systems and practically no antibodies.  How do microbes in the gut (gastro-intestinal tract) stimulate immunity?  This group looked at the specificity of antibodies that develop after GF mice are colonized by individual bacterial species and strains (monocolonization).  They used 8 strains of bacteria to inoculate GF mice and, after 3 weeks, analyzed the specificity of antibodies produced in the gut (recovered from fecal matter) and blood, focusing on the IgA isotype that protects mucosal surfaces.  

Not surprisingly, the antibodies tend to bind specifically to those strains of microbes against which they were stimulated (shown, Figure 1, panel A). They also found that mice monocolonized from birth produce more IgA reactive with that species (termed ‘self’) than newly-introduced species. They make a point about IgA being able to ‘aggregate pathogenic bacteria’ and ‘selectively coat disease-associated bacteria’ but it is unclear how IgA itself could distinguish dangerous from benign and anyway they tested only benign bacteria.  

A small panel (29) of monoclonal IgA antibodies cloned from gut tissues of monocolonized mice also showed species specificity. Finally, they showed that monoclonal IgA antibodies with specific binding activity could be detected in the feces of mice that had been force-fed the IgA 3 hours previously, suggesting a targeted, potential therapy (e.g., against the human pathogen Clostridioides difficile). 

Yang C, Chen-Liaw A, Spindler MP, Tortorella D, Moran TM, Cerutti A, Faith JJ. Immunoglobulin A antibody composition is sculpted to bind the self gut microbiome. ScienceImmunology. 2022 Jul 15;7(73) 

Wednesday, September 21, 2022

Covid Vax vs Variants

The first RNA vaccines against Covid were based on the Surface, S or “Spike” protein from the reference genome of SARS-CoV-2, Wuhan-Hu-1, published in January 2020.  Since then, more-infectious variants have emerged; one, D614G, dominated but did not escape vaccine protection probably because its characteristic mutation is located outside the Receptor Binding Domain (RBD, 319-541). In the past year, delta variants were overtaken by omicron variants, which have numerous changes in the RBD, raising concern that they escape current RNA vaccines. 

Neutralization assays measure the ability of blood-borne antibodies to block viral infection of cells grown in a culture dish; they are thought to provide valid measurements of protection. These authors previously tested Covid neutralization with blood sera from 15 health care workers, 4 vaccinated with Moderna (mRNA-1273) and 11 with Pfizer-BioNTech (BNT162b2). They confirmed that a third dose (second booster) increased the neutralization activity (titer) against all strains, albeit 3-4 times weaker against subvariants compared with the ancestral version. Moreover, they showed the neutralization titers induced by 3 doses of the vaccines, and therefore presumably levels of protection provided, approximated those found in convalescent (sick) Covid patients (Fig 1 panel B vs C and D). 

In this letter, they studied 46 health care workers, 24 vaccinated and boosted with Moderna and 22 with Pfizer-BioNTech. Fourteen of the cohort were infected during the follow-up year. Figure S3 in the supplementary data confirm the protection provided by a third dose (second booster). They found that neutralization titers against all strains declined over time.  Titers from vaccinated people remained within a substantial fraction of those from infected people (shown, dashed vs solid lines). 

How much protection -- neutralization titer -- is enough? A paper published last year analyzing 7 different vaccines reported that neutralization titers are predictive of protection.  Neutralization titers of 20% of convalescent levels protected on average half of people against detectable infection; levels at 3% convalescent protected against severe Covid. Taken together, these results seem to support the value of the ‘old’ vaccines against the newer variants. 

Qu P, Faraone JN, Evans JP, Zheng YM, Yu L, Ma Q, Carlin C, Lozanski G, Saif LJ, Oltz EM, Gumina RJ, Liu SL. Durability of Booster mRNA Vaccine against SARS-CoV-2 BA.2.12.1, BA.4, and BA.5 Subvariants. N Engl J Med. 2022 Sep 7. 

Tuesday, September 13, 2022

Obesity and severe COVID

Early in the COVID-19 pandemic, it was recognized that obesity seemed to be related to respiratory failure, or severe acute respiratory syndrome (SARS). These researchers tested whether leptin, a cytokine produced by fat cells in the gut and working on brain cells to influence hunger, might be correlated with risk, perhaps a biomarker of risk. 

They compared 31 obese COVID patients on ventilators with 8 non-infected, non-obese critically ill patients.  They found much higher levels of leptin in the patients with COVID (averages 21 vs 6 ug/l, with very good statistical significance, p = .0007).  The individual measurements overlap (shown), so leptin is not ‘the’ biomarker but clearly related. Whether related as a cause or consequence only a prospective trial could test rigorously. 

Figure 2 detail: BMI of patients panel A and Leptin levels panel B.  In each panel, ‘control’ critically ill patients, left, COVID patients right. 


The authors hypothesized that elevated leptin causes a ‘hyper immune’ state, especially stimulating lung epithelial cells.  They noted similar observation previously published for influenza and MERS. Since this paper was published, several groups have reported similar findings 2020 paper that was largely replicated (see review, which cites 3 later papers).  Ironically, shutdowns that have been effective in protecting many people from infection have also increased sedentary lifestyles, BMI, and risk. 


van der Voort PHJ, Moser J, Zandstra DF, Muller Kobold AC, Knoester M, Calkhoven CF, Hamming I, van Meurs M. Leptin levels in SARS-CoV-2 infection related respiratory failure: A cross-sectional study and a pathophysiological framework on the role of fat tissue. Heliyon. 2020 Aug;6(8):

Wednesday, September 7, 2022

A kidney ‘punch’ can tell much

renal biopsy is when a small piece of a patient's kidney is removed for analysis, usually by inserting a needle through the skin and into the kidney. Though invasive, the clinical value can be high for helping decide how to treat lupus or transplanted patients.  How to maximize that value? 

In this study, Clark and colleagues applied computer imaging techniques to answer why only about half of lupus patients with inflamed kidneys (nephritis) proceed to kidney failure (end stage renal disease, ESRD) and lose their kidney. They supposed there were subtle differences in the “frequency and organization of principal cellular effectors” between those patients who did, or didn’t, progress. They obtained biopsies from a cohort of 55 well-characterized lupus patients, of which 19 progressed to ESRD. They labeled very thin slices (sections) of kidney with 6 markers (CD3, CD4, CD20, CD11c, BDCA2, and DAPI) and counted several types of immune cells using confocal microscopy and deep learning analysis. 

Figure 2: left panel (H) are CD20+ B cells, right panel (I are CD3+CD4- T cells, (probably CD8+?).

They found a remarkably clear distinction: those with few B cells and many CD4- T cells, probably CD8+, often progress to ESRD (shown in red, panels H and I from figure 2).  Patients with many B cells and few CD4- T cells do not progress to ESRD (blue).  In the discussion, the authors mention that some clinical trials have targeted exactly those cells that this study indicate may be protective (B cells) or innocuous (CD4+ T cells).  A validated "identification, friend or foe" (IFF) system seems a good principle before aiming and firing.  

Abraham R, Durkee MS, Ai J, Veselits M, Casella G, Asano Y, Chang A, Ko K, Oshinsky C, Peninger E, Giger ML, Clark MR. Specific in situ inflammatory states associate with progression to renal failure in lupus nephritis. J Clin Invest. 2022 Jul 1;132(13)

Sunday, February 21, 2016

Skinny Genes: Human Warming and the Reverse Butterfly Effect

Obesity in humans is associated with a region around FTO, a "fat mass and obesity-associated” gene on chromosome 16. Although this is the strongest genetic association, it accounts for only a 1-2% difference in Body Mass Index (BMI) and the mechanism was unknown. Here, the investigators looked at regulator proteins binding to variants DNA sequences within the FTO region, particularly those binding to sites with single nucleotide polymorphisms (SNPs), in 100 healthy Europeans -- 52 subjects were homozygous for 3 risk-variant SNPs (both alleles, all 3 loci) and the remaining 48 were homozygous for the non-risk variants.

They found that the change of a T-to-C at one SNP within a risk allele of FTO prevented the binding of the repressor protein ARID5B. For want of this binding site, the repressor is lost. For want of this repressor, the expression of 2 linked genes doubles: IRX3, located about half a million base pairs (~0.5 Mbp) away, and IRX5, ~1 Mbp away. IRX3 repression made mice thinner by “increased energy dissipation without a change in physical activity or appetite”, i.e. not changing eating or exercise but rather elevating ‘metabolism’. The doubled expression of IRX3 led to a 5-fold reduction in mitochondrial thermogenesis and a 7-fold difference in brown/white adipose tissue development.  (Brown fat is brown because it holds more mitochondria, little furnaces that burn fat and produce heat.)

The risk alleles are causative, and mediate through IRX3 and IRX5, because mimicking the repression of IRX3 or IRX5 in 8 carriers of the risk alleles, but not 10 carriers of the non-risk allele increased stimulated metabolism (Fig 3D, shown, left panel). And overexpression of IRX3 or IRX5 reduced stimulated metabolism in non-risk allele carriers (because their endogenous genes are repressed) but not in risk allele carriers (Fig 3D, right panel).

Figure 3D. Oxygen consumption rate (OCR), basal and stimulated, in cells with risk or non-risk alleles.

The take-home messages are that the causative SNP can be kilobases away from the genes that mediate the effect (not a huge surprise) and that small risks might develop from relatively big differences in particular developmental and cell biological pathways (reverse butterfly effect: not small-change-to-big-effect but big effect leads to small change (1-2% BMI)).

N Engl J Med. 2015 Sep 3;373(10):895-907. “FTO Obesity Variant Circuitry and Adipocyte Browning in Humans.” Claussnitzer M, Dankel SN, Kim KH, Quon G, Meuleman W, Haugen C, Glunk V, Sousa IS, Beaudry JL, Puviindran V, Abdennur NA, Liu J, Svensson PA, Hsu YH, Drucker DJ, Mellgren G, Hui CC, Hauner H, Kellis M

Sunday, November 22, 2015

Normalizing T lymphocyte metabolism treats lupus autoimmunity

Glucose is metabolized in two pathways to fuel cellular functions: glycolysis, which splits glucose, yielding little energy but providing pyruvate and other materials for synthesis, and oxidative phosphorylation, which degrades glucose in the mitochondria and produces ~15-fold more energy. Glucose uptake is a limiting in activated T lymphocytes through CD28 costimulation.  Glucose metabolism is dysregulated in T lymphocytes of patients with the autoimmune disease Systemic Lupus Erythematosus (SLE, lupus, review).

These investigators blocked glycolysis with 2-deoxy-D-glucose (2DG) and oxidative phosphorylation with metformin (Met), and observed that disease was reduced and even reversed in mice “triple congenic” (TC) with three lupus-predisposing genetic regions: Sle1-Sle2-Sle3 (review).  2DG is glucose with its 2-hydroxl group replaced by a hydrogen, thereby blocking glycolysis.  Met is a small molecule that was discovered in 1920s to reduce blood glucose, probably by interfering with mitochondrial respiration.  The authors show here that Met reduces extracellular acidification rate (ECAR) and 2DG reduces oxygen consumption rate (OCR), both measures of glucose metabolism, in activated T cells (fig. 1).  

Anti-nuclear antibodies (ANA), a hallmark of lupus, are particularly dangerous because they damage glomeruli, the kidney’s filtration units, causing glomerular nephritis (GN).  The authors show a remarkable reduction in ANA and spleen size (fig. 4, panels, C, D and a portion of panel E shown here) as well as improvement in kidney pathology (fig. 4 panel I)
Although these metabolism inhibitors are not targeted to pathogenic T cells, there are no obvious adverse consequences for the animal or even the immune system.  Treated mice raise antibody responses following protein immunization, generating normal levels and avidities of circulating antibodies (supplemental).  Perhaps the limiting effect of glucose uptake by pathogenic, chronically activated T cells make them more sensitive to inhibition.  How treatment influences control of chronic infections (e.g., EBV, CMV) is also worth knowing.  There was no change in body weight on Met. 
Testing 2 other mouse models of lupus (NZB/W and chronic graft-vs-host (cGVH)), they found a mixture of responses.  For example, in cGVH, combined treatment doesn’t reduce spleens (though Met alone does), while treatment of NZB/W mice reduces ANA but doesn’t improve GN.  Human patients exhibit a range of symptoms and might also be expected to show a range of responses.  This is inspired and inspiring work that cuts across as many disciplines as it does organ systems and raises as many questions as hopes. 
Y. Yin, S.-C. Choi, Z. Xu, D. J. Perry, H. Seay, B. P. Croker, E. S. Sobel, T. M. Brusko, L. Morel, Normalization of CD4+ T cell metabolism reverses lupus. Sci. Transl. Med. 7, 274ra18 (2015).

Sunday, March 15, 2015

Stem Cells: Unstable in Culture

Stem Cells (SC) can differentiate into many different cell types, offering the potential of replacing failing cells, tissues, or even entire organs with new ones generated from the patient’s own or related donor SC (National Academy of Sciences Workshop summary).  To be useful for therapy in the clinic, it would be necessary to grow and expand SCs in culture.

The authors explored the proliferation of human embryonic SC (HESC), which are prepared from disrupted embryos, and the less-controversial human inducible pluripotent stem cells (hiPSCs), which can be prepared from several adult tissues, including blood, skin, and fat.  They obtained 1 HESC line, WA09, from the WiCell Research Institute and they generated 3 hiPSC lines from fetal dermal fibroblasts by over-expressing the ‘standard reprogramming factors’ (pluripotency-conferring genes, transduced OCT4/POU5F1, SOX2, KLF4, and MYC).

They compared four standard SC culture conditions: with or without a feeder cell layer and enzymatic or “mechanical” (dissection) disruption, with 6 replicate cultures per condition, for over 100 “passages” (transfers to fresh cultures). Previous studies cited here revealed genomic changes (small duplications) that are not detectable by karyotyping, particularly on chromosome 12, where the pluripotency-related gene NANOG is encoded, and chromosome 20, where the survival gene Bcl-xL is encoded.  In addition to measuring proliferation, telomere length, pluripotency by teratoma formation, they also analyzed over a million reference SNPs around the genome and used those SNPs to assess copy number variation (CNV). 

Not surprisingly, genomic changes increased with time in culture, both in aberration number (A) and total length (B) (Figure 2, shown, WA09 HESC: left duplications and right deletions).  The number of aberrations was lowest in “EcmMech” condition, i.e. cultures without feeder cells (only extracellular matrix, ECM), and disrupted mechanically (blue line).  The number and length of aberrations was worst with MefEnz (green line), cultured with feeder cells (mouse embryo fibroblasts, Mef) and disrupted enzymatically.  They conclude that there is a “need for careful assessment of the effects of culture conditions on cells intended for clinical therapies”.  

Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions  Garitaonandia et al. PLoS One 10(2), February 25, 2015

Saturday, January 24, 2015

Nurture Immunity: Immune system influenced more by environment than by genes

Differences in immune protection presumably explain why some people exposed to infection resist disease or recover while others succumb.  These authors sought to distinguish the influences of genes and environment on immunity. They compared the cellular and molecular components of the immune system among 210 twins: 78 monozygotic (MZ, “identical”) and 27 dizygotic (DZ, fraternal) pairs.  They measured 43 serum proteins and 72 immune cell populations repeatedly and longitudinally (over time) to assess actual variations and account for technical variations.  MZ twins, who have practically identical genomes, and DZ twins, who share half their genes, are especially valuable for assessing the relative contributions of “nature or nurture” (genes or environment) to phenotype.  Their analysis allowed them to detect as little as 20% heritability.

The levels of few proteins and cell populations are under strong genetic control, such as interleukin-6 and CD4+ “central memory” T cells, but most are only weakly heritable or not at all (Fig 1). They found that a common, chronic infection, by cytomegalovirus (CMV), influences the levels of most (58%) cell populations and proteins (Fig 5).  Variation between twins increased as they age, probably reflecting different environmental stimuli and epigenetic changes (Fig 4).  Most intriguing, they correlate the heritability of response to vaccines to the age of immunization, whereby early childhood vaccines are highly heritable while vaccines after early adolescence have no detectable heritability (Table 1, shown below).  

Brodin et al. Cell. 2015 Jan 15;160(1-2):37-47. Variation inthe human immune system is largely driven by non-heritable influences. 

Sunday, December 8, 2013

Levels of a common, chronic virus (TTV) reflects the immune competence of transplant recipients

Successful organ transplantation requires careful immune suppression: enough to block the rejection of transplant while permitting host defense against infectious microbes.   Viruses that are not cleared by our immune systems, are common in healthy people, and can complicate transplantation include cytomegalovirus (CMV) and Torque teno virus (TTV), which was first described in 1997 [review].  TTV is a small (3.8 kb), single-stranded, transfusion transmitted DNA virus, representative of a highly diverse family of anelloviruses 

The authors examined the influence of immune-suppressive drugs (e.g., tacrolimus, mycophenolate mofitil, cyclosporine) and the anti-CMV drug valgancyclovir on chronic, endogenous microbes.  From 96 heart or lung transplant recipients they collected 656 blood samples over time, some up to a year post-transplant, removed the cells, and identified remaining DNA by sequencing.  They found that 0.12% matched viral or bacterial or fungal sequences.  They validated some ‘hits’ with quantitative PCR.  Control preparations using water or bacteriophage demonstrated no relevant artifacts or contamination.  

They found that treatment with valgancyclovir reduced herpesviruses, including CMV, but dramatically increased the relative and absolute levels of anelloviruses, including TTVs (fig. 2, 3, 4).   Moreover, those patients who did not reject their transplants tended to have a greater increase in anelloviruses (Fig. 5A, shown; rejecting patients plotted in red).  The authors conclude that anellovirus levels might be used to monitor immune competence.   Focosi et al. made a related observation following autologous stem cell transplantation

 Cell. 2013 Nov 21;155(5):1178-87. Temporal response of the human virome to immunosuppression and antiviral therapy.  De Vlaminck I, Khush KK, Strehl C, Kohli B, Luikart H, Neff NF, Okamoto J, Snyder TM, Cornfield DN, Nicolls MR, Weill D, BernsteinD, Valantine HA, Quake SR. 

Sunday, October 27, 2013

Salt develops a taste for Th17 lymphocytes

Helper T lymphocytes that make the hormone interleukin-17 (IL-17), called Th17 cells, contribute to inflammation and autoimmune diseases (review). The development of Th17 cells was known to require IL-23 but it was not known exactly why.  To gain some perspective, the authors measured gene transcripts found in Th17 cells as they develop over time from na├»ve mouse T lymphocytes treated with transforming growth factor-beta (TGFb) and IL-6.  They found that SGK1, an enzyme that phosphorylates proteins and has been shown to regulate sodium (Na+) transport and salt (NaCl) balance in other cells, was induced nearly 200-fold.  They emphasize that IL-23 is “critical” to the induction and maintenance of SGK1 but much of that evidence is relegated to the supplement data.  “Network analysis” with a computer program strengthened their suspicion that SGK1 is a “node” in the IL-23 signaling pathway.  
Mice without SGK1 (SGK1-knockouts, KO) have fewer Th17 cells that make less IL-17 when treated with IL-23; notably SGK1 deficiency also alters genes regulating other T cell subsets, including interferon-gamma (Ifng), Tbx21, and Gata3 (see also the previous gloss on transcription factors regulating Th17).  To test the role of SGK1, they immunized “floxed” SGK1 (conditional KO) mice with a myelin protein (MOG), which induces in some mouse strains a multiple sclerosis (MS) like disease called experimental autoimmune encephalitis (EAE).  EAE severity was significantly reduced in mice without SGK1 in Th17 cells or CD4+ helper T cells,  (fig 2a, KO score <1 normal="" vs.="">3), which corresponded with a greatly reduced number of Th17 cells in the organ targeted by this autoimmune disease, the central nervous system (CNS).  They also saw that CNS-infiltrating cells in EAE had expressed IL-17 at one time (eYFP+, fig 2e) but that expression of IL-17 by CD4 cells was lost in SGK1-KO animals (eYFP+ IL17-), suggesting that SGK1 was required to maintain expression.
That was nice but now the spice – could dietary salt modulate immunity through SGK1?    Indeed, they found that a high salt diet (HSD) accelerates the development of EAE in normal mice (fig 4e, top 2 lines, trend line offset to the left is HSD) while it does nothing to the GSK1-KO animals (lower, lines).  And connecting at least one of the dots between diet and Th17, they found that HSD also increased more than 2-fold Th17 cells and to a lesser degree interferon-gamma expressing cells in the CNS,  and the induction depended on SGK1 (fig 5f, shown, Th17 left, IFNg right; open bars SGK1-CD4-KO).  A companion paper pursued the role of dietary salt in EAE   

Nature. 2013 Apr 25;496(7446):513-7.  Induction of pathogenic TH17 cells by inducible salt-sensing kinase SGK1.  Wu C, Yosef N, Thalhamer T, Zhu C, Xiao S, Kishi Y, Regev A, Kuchroo VK.