Medical students' perceptions of an artificial intelligence (AI) assisted diagnosing program.

As artificial intelligence (AI) assisted diagnosing systems become accessible and user-friendly, evaluating how first-year medical students perceive such systems holds substantial importance in medical education. This study aimed to assess medical students' perceptions of an AI-assisted diagnostic tool known as 'Glass AI.' Data was collected from first year medical students enrolled in a 1.5-week Cell Physiology pre-clerkship unit. Students voluntarily participated in an activity that involved implementation of Glass AI to solve a clinical case. A questionnaire was designed using 3 domains: 1) immediate experience with Glass AI, 2) potential for Glass AI utilization in medical education, and 3) student deliberations of AI-assisted diagnostic systems for future healthcare environments. 73/202 (36.10%) of students completed the survey. 96% of the participants noted that Glass AI increased confidence in the diagnosis, 43% thought Glass AI lacked sufficient explanation, and 68% expressed risk concerns for the physician workforce. Students expressed future positive outlooks involving AI-assisted diagnosing systems in healthcare, provided strict regulations, are set to protect patient privacy and safety, address legal liability, remove system biases, and improve quality of patient care. In conclusion, first year medical students are aware that AI will play a role in their careers as students and future physicians.

Inflammatory immune cells may impair the preBCR checkpoint, reduce new B cell production, and alter the antibody repertoire in old age.

Aging impairs development of new B cells and diminishes the expression of protective antibodies. Reduced numbers of B cell precursors generally occur in old (~2 yrs.) mice. At the pro-B to pre-B cell transition, the pre-B cell receptor (preBCR) checkpoint directs pre-B cell expansion and selection of the pre-B cell immunoglobulin (Ig) µ heavy chain variable region repertoire. The preBCR is comprised of Ig µ heavy chain + surrogate light chains (SLC; λ5/VpreB). In old B cell precursors, SLC is decreased and fewer pre-B cells form the preBCR. In pro-B cells, SLC is complexed with cadherin 17 to form a "pro-B cell receptor" whose signaling is postulated to increase apoptotic sensitivity. We propose that inflammation in old mice, in part mediated by the age-associated B cells (ABC), promotes apoptosis among pro-B cells, particularly those relatively high in SLC. The remaining pro-B cells, with lower SLC, now generate pre-B cells with limited capacity to form the preBCR. Ig µ heavy chains vary in their capacity to associate with SLC and form the preBCR. We speculate that limited SLC restricts formation of the preBCR to a subset of Ig µ heavy chains. This likely impacts the composition of the antibody repertoire among B cells.

Age-associated B cells (ABC) inhibit B lymphopoiesis and alter antibody repertoires in old age.

With old age (∼2y old), mice show substantial differences in B cell composition within the lymphoid tissues. In particular, a novel subset of IgM+ CD21/35lo/- CD23- mature B cells, the age-associated B cells or ABC, increases numerically and proportionately. This occurs at the expense of other B cell subsets, including B2 follicular B cells in spleen and recirculating primary B cells in bone marrow. Our studies suggest that ABC have a distinctive antibody repertoire, as evidenced by relatively high reactivity to the self-antigens phosphorylcholine (PC) and malondialdehyde (MDA). While PC and MDA are found on apoptotic cells and oxidized lipoproteins, antibodies to these antigens are also cross-reactive with epitopes on bacterial species. In old mice, ABC express TNFα and are pro-inflammatory. ABC can inhibit growth and/or survival in pro-B cells as well as common lymphoid progenitors (CLP). In particular, ABC cause apoptosis in pro-B cells with relatively high levels of the surrogate light chain (SLC) and, consequently, promote an "SLC low" pathway of B cell differentiation in old mice. SLC together with µ heavy chain comprises the pre-B cell receptor (preBCR) critical for pre-B cell expansion and selection of the µ heavy chain Vh repertoire. The low level of SLC likely impairs normal preBCR driven proliferation and alters µ heavy chain Vh selection thereby affecting the antibody specificities of new B cells. In this manner, ABC may contribute to both qualitative and quantitative disruptions of normal B lymphopoiesis in old age.

In old BALB/c mice, bone marrow pre-B cell and surrogate light chain reduction is associated with increased B cell reactivity to phosphorylcholine, but reduced T15 idiotype dominance.

In young adult BALB/c mice, antibodies to phosphorylcholine (PC) bearing the T15 (TEPC 15) idiotype confer protection against pneumococcal infections. In old age, even though PC reactive B cells are often increased, the proportion of T15+ antibodies declines. We hypothesize that limited surrogate light chain (SLC) and compromise of the pre-B cell receptor checkpoint in old mice contribute to both reduced new B cell generation and changes in the anti-PC antibodies seen in old age. In old mice: 1) early pre-B cell loss is most pronounced at the preBCR checkpoint; however, the reduced pool of early pre-B cells continues to proliferate consistent with preBCR signaling; 2) increased PC reactivity is seen in bone marrow immature B cells; 3) deficient SLC promotes increased B cell PC reactivity and diminished T15 idiotype expression; and 4) as pre-B cell losses and reduced SLC become progressively more severe, increased T15 negative PC reactive B cells occur. These results associate a reduction in pre-B cells, imposed at the preBCR checkpoint, with increased reactivity to PC, but more limited expression of the protective T15 idiotype among PC reactive antibodies in old age.

In aged mice, low surrogate light chain promotes pro-B-cell apoptotic resistance, compromises the PreBCR checkpoint, and favors generation of autoreactive, phosphorylcholine-specific B cells.

In aged mice, new B-cell development is diminished and the antibody repertoire becomes more autoreactive. Our studies suggest that (i) apoptosis contributes to reduced B lymphopoiesis in old age and preferentially eliminates those B-cell precursors with higher levels of the surrogate light chain (SLC) proteins (λ5/VpreB) and (ii) λ5(low) B-cell precursors generate new B cells which show increased reactivity to the self-antigen/bacterial antigen phosphorylcholine (PC). Pro-B cells in old bone marrow as well as pro-B cells from young adult λ5-deficient mice are resistant to cytokine-induced apoptosis (TNFα; TGFß), indicating that low λ5 expression in pro-B cells is sufficient to cause increased survival. Transfer of TNFα-producing 'age-associated B cells' (ABC; CD21/35(-) CD23(-)) or follicular (FO) B cells from aged mice into RAG-2 KO recipients led to preferential loss of λ5(high) pro-B cells, but retention of λ5(low), apoptosis-resistant pro-B cells. In old mice, there is increased reactivity to PC in both immature bone marrow B cells and mature splenic FO B cells. In young mice, absence of λ5 expression led to a similar increase in PC reactivity among bone marrow and splenic B cells. We propose that in old age, increased apoptosis, mediated in part by TNFα-producing B cells, results in preferential loss of SLC(high) pro-B cells within the bone marrow. Further B-cell development then occurs via an 'SLC(low)' pathway that not only impairs B-cell generation, but promotes autoreactivity within the naïve antibody repertoires in the bone marrow and periphery.

Impaired B lymphopoiesis in old age: a role for inflammatory B cells?

Continued generation of new B cells within the bone marrow is required throughout life. However, in old age, B lymphopoiesis is inhibited at multiple developmental stages from hematopoietic stem cells through the late stages of new B cell generation. While changes in B cell precursor subsets, as well as alterations in the supporting bone marrow microenvironment, in old age have been known for the last 20 years, only more recently have insights into the cellular and molecular mechanisms responsible become clarified. Our recent discovery that B cells in aged mice are pro-inflammatory and can diminish B cell generation within the bone marrow suggests a potential mechanism of inappropriate "B cell feedback" which contributes to a bone marrow microenvironment unfavorable to B lymphopoiesis. We hypothesize that the consequences of a pro-inflammatory microenvironment in old age are (1) reduced B cell generation and (2) alteration in the "read-out" of the antibody repertoire. Both of these likely ensue from reduced expression of the surrogate light chain (λ5 + VpreB) and consequently reduced expression of the pre-B cell receptor (preBCR), critical to pre-B cell expansion and Vh selection. In old age, B cell development may progressively be diverted into a preBCR-compromised pathway. These abnormalities in B lymphopoiesis likely contribute to the poor humoral immunity seen in old age.

A comparative review of aging and B cell function in mice and humans.

Immune system function declines with age. Here we review and compare age-associated changes in murine and human B cell pools and humoral immune responses. We summarize changes in B cell generation and homeostasis, as well as notable changes at the subcellular level; then discuss how these changes help to explain alterations in immune responses across the adult lifespan of the animal. In each section we compare and contrast findings in the mouse, arguably the best animal model of the aging immune system, with current understanding of B cell immunity in humans.

In senescence, age-associated B cells secrete TNFα and inhibit survival of B-cell precursors.

Aged mice exhibit ~ 5-10-fold increases in an ordinarily minor CD21/35(-) CD23(-) mature B-cell subset termed age-associated B cells (ABCs). ABCs from old, but not young, mice induce apoptosis in pro-B cells directly through secretion of TNFα. In addition, aged ABCs, via TNFα, stimulate bone marrow cells to suppress pro-B-cell growth. ABC effects can be prevented by the anti-inflammatory cytokine IL-10. Notably, CD21/35(+) CD23(+) follicular (FO) splenic and FO-like recirculating bone marrow B cells in both young and aged mice contain a subpopulation that produces IL-10. Unlike young adult FO B cells, old FO B cells also produce TNFα; however, secretion of IL-10 within this B-cell population ameliorates the TNFα-mediated effects on B-cell precursors. Loss of B-cell precursors in the bone marrow of old mice in vivo was significantly associated with increased ABC relative to recirculating FO-like B cells. Adoptive transfer of aged ABC into RAG-2 KO recipients resulted in significant losses of pro-B cells within the bone marrow. These results suggest that alterations in B-cell composition during old age, in particular, the increase in ABC within the B-cell compartments, contribute to a pro-inflammatory environment within the bone marrow. This provides a mechanism of inappropriate B-cell 'feedback' that promotes down-regulation of B lymphopoiesis in old age.

A molecular mechanism for TNF-α-mediated downregulation of B cell responses.

B cell function with age is decreased in class switch recombination (CSR), activation-induced cytidine deaminase (AID), and stability of E47 mRNA. The latter is regulated, at least in part, by tristetraprolin (TTP), which is increased in aged B cells and also negatively regulates TNF-α. In this study, we investigated whether B cells produce TNF-α, whether this changes with age, and how this affects their function upon stimulation. Our hypothesis is that in aging there is a feedback mechanism of autocrine inflammatory cytokines (TNF-α) that lowers the expression of AID and CSR. Our results showed that unstimulated B cells from old BALB/c mice make significantly more TNF-α mRNA and protein than do B cells from young mice, but after stimulation the old make less than the young; thus, they are refractory to stimulation. The increase in TNF-α made by old B cells is primarily due to follicular, but not minor, subsets of B cells. Incubation of B cells with TNF-α before LPS stimulation decreased both young and old B cell responses. Importantly, B cell function was restored by adding anti-TNF-α Ab to cultured B cells. To address a molecular mechanism, we found that incubation of B cells with TNF-α before LPS stimulation induced TTP, a physiological regulator of mRNA stability of the transcription factor E47, which is crucial for CSR. Finally, anti-TNF-α given in vivo increased B cell function in old, but not in young, follicular B cells. These results suggest new molecular mechanisms that contribute to reduced Ab responses in aging.

E47 retroviral rescue of intrinsic B-cell defects in senescent mice.

In aging, immune responses are dramatically impaired, specifically the ability to produce protective antibodies. We previously showed that with age there is a B-cell intrinsic decrease in class switch recombination (CSR) because of a decrease in activation-induced cytidine deaminase (AID). One mechanism we have demonstrated for decreased AID includes increased mRNA degradation of the transcription factor E47, critical for AID transcription. Here, we show by means of a retroviral construct containing the DsRED reporter and the 3'UTR of E47 that the 3'UTR lowers mRNA expression, and particularly in B cells from old mice. This is the first demonstration that the E47 3'UTR directly regulates its degradation. The AID mRNA was not differentially regulated by degradation in aging. Therefore, we have here further established critical components for decreased AID with age. The major aim of this study was to establish conditions for the rescue of the intrinsic defect of aged B cells with retroviral addition of the coding region of E47 in splenic B cells to restore their ability to produce optimal AID and class switch to IgG. In this study, we show that young and old primary B cells overexpressing a stable E47 mRNA up-regulate E47, AID, and CSR and improve B-cell immune responses in senescent murine B cells. Our results provide a proof of principle for the rescue of intrinsic B-cell defects and the humoral immune response in senescence.

Protein phosphatase 2A (PP2A) is increased in old murine B cells and mediates p38 MAPK/tristetraprolin dephosphorylation and E47 mRNA instability.

The transcription factor E47, which regulates immunoglobulin class switch in murine splenic B cells, is down-regulated in aged B cells due to reduced mRNA stability. Part of the decreased stability of E47 mRNA is mediated by tristetraprolin (TTP), a physiological regulator of mRNA stability. We have previously shown that TTP mRNA and protein expression are higher in old B cells, and the protein is less phosphorylated in old B cells, both of which lead to more binding of TTP to the 3'-UTR of E47 mRNA, thereby decreasing its stability. PP2A is a protein phosphatase that plays an important role in the regulation of a number of major signaling pathways. Herein we show that not only the amount but also the activity of PP2A is increased in old B cells. As a consequence of this higher phosphatase activity in old B cells, p38 MAPK and TTP (either directly or indirectly by PP2A) are less phosphorylated as compared with young B cells. PP2A dephosphorylation of p38 MAPK and/or TTP likely generates more binding of the hypophosphorylated TTP to the E47 mRNA, inducing its degradation. This mechanism may be at least in part responsible for the age-related decrease in class switch.

B cells and aging: molecules and mechanisms.

Recent advances allow aging-associated changes in B-cell function to be approached at a mechanistic level. Reduced expression of genes crucial to lineage commitment and differentiation yield diminished B-cell production. Moreover, intrinsic differences in the repertoire generated by B-cell precursors in aged individuals, coupled with falling B-cell generation rates and life-long homeostatic competition, result in narrowed clonotypic diversity. Similarly, reductions in gene products crucial for immunoglobulin class switch recombination and somatic hypermutation impact the efficacy of humoral immune responses. Together, these findings set the stage for integrated analyses of how age-related changes at the molecular, cellular and population levels interact to yield the overall aging phenotype.

Effects of fenbendazole on the murine humoral immune system.

Pinworms are highly contagious parasites that have been effectively treated in laboratory rodents with fenbendazole (FBZ). Whether FBZ has any detrimental side effects that may compromise experimental results is unknown. Here we asked whether the immune systems from young and aged mice are altered under FBZ treatment. We compared control and FBZ-treated groups of young (age, 2 to 4 mo) and old (age, 22 to 24 mo) BALB/cN mice. The treated mice received a total of 4 wk (alternating-week treatment regimen) of FBZ-medicated feed. Spleen and bone marrow were collected for immunologic assays, and heart, stomach, intestines, kidneys, and liver were evaluated by histopathology. Our results indicate that FBZ treatment has significant effects on the immune systems of mice; these effects are greater in aged mice. FBZ treatment adversely affected mRNA and protein expression of E2A (a transcription factor crucial for B lymphocytes) in activated precursor B lymphocytes obtained from the bone marrow of young and old mice. These effects were reversed by 6 wk on regular feed after the end of treatment. Activated B lymphocytes from the spleens of young and old mice showed decreased function (cell proliferation, E2A mRNA and protein expression) through the last time point of FBZ treatment but recovered by 2 to 4 wk after treatment. Our findings suggest that FBZ treatment may alter sensitive immune and molecular measures as presented here, and postponing the experimental use of mice until at least 6 wk after treatment should be considered.

NK cells in the CD19- B220+ bone marrow fraction are increased in senescence and reduce E2A and surrogate light chain proteins in B cell precursors.

E2A encoded proteins, key transcriptional regulators in B lineage specification and commitment, have been shown to decrease in B cell precursors in old age. E2A regulates genes encoding the surrogate light chain proteins lambda5 and VpreB. In old age, B cell precursors express less surrogate light chain and this results in compromised pre-B cell receptor function and diminished expansion of new pre-B cells in senescence. Herein, we show that aged bone marrow has increased Hardy Fraction A (CD19(-) B220(+)) cells, including NK cells, that can inhibit both E47 (E2A) protein and surrogate light chain protein expression in B cell precursors. In vitro, NK-associated inhibition of E47 protein is contact-independent and partially reversed by neutralization of TNFalpha. In vivo, depletion of NK cells in aged mice by treatment with anti-asialo GM1 antibody led to restoration of surrogate light chain protein levels to that typical of young B cell precursors. These studies suggest that NK cells, within the CD19(-) B220(+) bone marrow cell fraction, may contribute to a bone marrow microenvironment that has the potential to negatively regulate E47 (E2A) as well as surrogate light chain levels in B cell precursors in old age.

Old mice retain bone marrow B1 progenitors, but lose B2 precursors, and exhibit altered immature B cell phenotype and light chain usage.

The bone marrow of old adult mice ( approximately 2 years old) has reduced B lymphopoiesis; however, whether the B1 pathway in adult bone marrow is also compromised in senescence is not known. Herein, we show that phenotypic (IgM(-)Lin(-)CD93(+)[AA4.1(+)] CD19(+)B220(low/-)) B1 progenitors are retained in old bone marrow even as B2 B cell precursors are reduced. Moreover, B1 progenitors from both young adult and old mice generated new B cells in vitro enriched for CD43 expression, likely due to their activation, and exhibited increased lambda light chain usage and diminished levels of kappa light chain expression. B1 progenitors were shown to have lower surrogate light chain (lambda5) protein levels than did B2 pro-B cells in young mice and these levels decreased in both B1 and B2 precursor pools in old age. These results indicate that the B1 B cell pathway persists during old age in contrast to the B2 pathway. Moreover, B1 B cell progenitors generated new B cells in the adult bone marrow that have distinct surface phenotype and light chain usage. This is associated with decreased surrogate light chain expression, a characteristic held in common by B1 progenitors as well as B2 precursors in old mice.

Deviation of the B cell pathway in senescent mice is associated with reduced surrogate light chain expression and altered immature B cell generation, phenotype, and light chain expression.

B lymphopoiesis in aged mice is characterized by reduced B cell precursors and an altered Ab repertoire. This likely results, in part, from reduced surrogate L chains in senescent B cell precursors and compromised pre-BCR checkpoints. Herein, we show that aged mice maintain an ordinarily minor pool of early c-kit(+) pre-B cells, indicative of poor pre-BCR expression, even as pre-BCR competent early pre-B cells are significantly reduced. Therefore, in aged mice, B2 B lymphopoiesis shifts from dependency on pre-BCR expansion and selection to more pre-BCR-deficient pathways. B2 c-kit(+) B cell precursors, from either young or aged mice, generate new B cells in vitro that are biased to larger size, higher levels of CD43, and decreased kappa L chain expression. Notably, immature B cells in aged bone marrow exhibit a similar phenotype in vivo. We hypothesize that reduced surrogate L chain expression contributes to decreased pre-B cells in aged mice. The B2 pathway is partially blocked with limited B cell development and reduced pre-BCR expression and signaling. In old age, B2 pathways have limited surrogate L chain and increasingly generate new B cells with altered phenotype and L chain expression.

Aging down-regulates the transcription factor E2A, activation-induced cytidine deaminase, and Ig class switch in human B cells.

Elderly humans have compromised humoral and cellular immune responses, which lead to reduced protection to infectious agents and to vaccines. Currently, available vaccines suboptimally protect the elderly population. The capacity to class switch the Ig H chain is critical to the effectiveness of humoral immune responses in mice and humans. We have previously shown in mice that the E2A-encoded transcription factor E47, which regulates many B cell functions, is down-regulated in old splenic B cells. This leads to a reduction in the activation-induced cytidine deaminase (AID), which is known to induce class switch recombination and Ig somatic hypermutation. The old activated murine B cells also have less AID and less switched Abs. We have extended our study here to investigate whether aging also affects Ab production and E47 and AID expression in B cells isolated from the peripheral blood of human subjects (18-86 years). Our results obtained with activated CD19(+) B cells show that the expression of E47, AID, and Iggamma1 circle transcripts progressively decrease with age. We also show an age-related decline in the percentage of switch memory B cells (IgG(+)/IgA(+)), an increase in that of naive B cells (IgG(-)/IgA(-)/CD27(-)) for most individuals, and no decrease in that of IgM memory cells in peripheral blood, consistent with our data on the decrease seen in class switch recombination in vitro. Our results provide a possible molecular mechanism for a B cell intrinsic defect in the humoral immune response with aging and suggest avenues for improvement of vaccine response in elderly humans.

Mechanisms for decreased function of B cells in aged mice and humans.

The immune system has been known for some time to be compromised in aged individuals, e.g., both mice and humans, and in both humoral and cellular responses. Our studies have begun to elucidate intrinsic B lymphocyte defects in Ig class switch recombination, activation-induced cytidine deaminase, and E47 transcription factor expression. These defects occur in both mice and humans. Our studies have also shown that tristetraprolin is one of the key players in regulating the decreased E47 mRNA stability in aged B lymphocytes. These and current studies should lead to improvements in B lymphocyte function in aged populations.

Tristetraprolin, a negative regulator of mRNA stability, is increased in old B cells and is involved in the degradation of E47 mRNA.

We have previously shown that the E2A-encoded transcription factor E47, which regulates class switch in splenic B cells, is down-regulated in old B cells, due to increased E47 mRNA decay. At least part of the decreased stability of E47 mRNA seen in aged B cells is mediated by proteins. We have herein looked at the specific proteins responsible for the degradation of the E47 mRNA and found that tristetraprolin (TTP), a physiological regulator of mRNA expression and stability, is involved in the degradation of the E47 mRNA. Although many studies have characterized TTP expression and function in macrophages, monocytes, mast cells, and T cells, little is known about the expression and function of TTP in primary B cells. We show herein that TTP mRNA and protein expression are induced by LPS in B cells from young and old mice, the levels of TTP in old B cells always being higher than those in young B cells. Although TTP mRNA is degraded at a significantly higher rate in old B cells, TTP mRNA expression is higher in old than in young, likely due to its increased transcription. Like in macrophages, TTP protein expression and function in B cells are dependent upon p38 MAPK. We found that there is less phospho-TTP (inactive form), as well as phospho-p38, in old than in young splenic-activated B cells. This is the first report showing that TTP is involved in the degradation of the E47 mRNA and is up-regulated in old B cells.

Accelerated Notch-dependent degradation of E47 proteins in aged B cell precursors is associated with increased ERK MAPK activation.

The transcriptional regulator E47, encoded by the E2A gene, is crucial to B lymphopoiesis. In BALB/c senescent mice (approximately 2 years old), the incidence of E47-expressing pro-B cells in vivo and E47 protein steady state levels in B cell precursors in vitro were reduced. Poor expression of E47 protein was a consequence of accelerated proteasome-mediated turnover and was associated with heightened ubiquitin modification of E2A-encoded proteins in aged B cell precursors. Both MAPK and Notch activity have been previously associated with E2A-encoded protein stability in lymphocytes. Aged B cell precursors exhibited heightened levels of MAPK activity reflected in increased levels of phospho-ERK proteins. Phosphorylation of E2A-encoded proteins was also increased in aged B cell precursors and pharmacologic inhibition of MEK-1 resulted in a partial restoration of their E47 protein. Both Notch proteins and their Delta-like ligands were detected comparably in young and aged B cell precursors. Either inhibition of Notch activation via gamma-secretase or Ab blockade of Notch-Delta-like ligand interactions partially restored E47 expression in aged B cell precursors. We hypothesize that increased MAPK activity promotes phosphorylation of E2A-encoded protein in aged B cell precursors. Subsequently, E2A-encoded proteins undergo ubiquitination and accelerated degradation in a Notch-dependent process. The dysregulation of E2A-encoded protein expression may contribute to the reductions seen in early B lymphopoiesis during murine senescence.