Enhancement of hematopoiesis and lymphopoiesis in diet-induced obese mice.

A rodent model of diet-induced obesity revealed that obesity significantly altered hematopoietic and lymphopoietic functions in the bone marrow and thymus. C57BL/6 mice were fed a mixed high-fat diet (HFD) of 45% fat or 10% fat diet (lean controls) for 180 d. A sustained increase in the numbers of cells found in bone marrow and thymus of HFD mice occurred from day 90 to day 180. However, with the exception of a 10-18% increase in the proportion of lymphocytes, the composition of monocytes, granulocytes, erythrocytes, and mixed progenitor lineages remained normal in the marrow. Likewise, thymuses of HFD mice increased 30-50% in size compared with controls, with analogous increases in thymocyte numbers. The overall thymus cellular composition remained normal. Although increased blood and lymphatic volume in obese mice would play a role in increased hematopoiesis, there were large and disproportionate increases in blood leukocytes of HFD mice, indicating that homeostasis was not maintained. Leptin, which promotes lymphopoiesis and myelopoiesis, reached 100 ng/mL in sera from HFD mice. Moreover, a three- to sixfold increase in adipocytes in marrow resulted in spiked leptin mRNA expression in bones of HFD mice compared with lean controls. Other cytokines and growth factors did not show any increases in obese marrow. The substantial increase in lymphopoietic and hematopoietic processes in HFD mice indicates that the primary tissues are another facet of the immune system dysregulated by obesity, which was perhaps fostered by higher amounts of leptin in marrow and serum.

Enhanced production of early lineages of monocytic and granulocytic cells in mice with colitis.

The bone marrow (BM) is a large, highly active, and responsive tissue. Interestingly, little is known about the impact of colitis on hematopoietic functions. Using dextran sodium sulfate (DSS) to induce colitis in mice, we identified significant changes in the BM. Specifically, cells of the monocytic and granulocytic lineages increased nearly 60% and 80%, respectively. This change would support and promote the large infiltration of the gut with neutrophils and monocytes that are the primary cause of inflammation and tissue damage during colitis. Conversely, the early lineages of B and T cells declined in the marrow and thymus with particularly large losses observed among pre-B and pre-T cells with heightened levels of apoptosis noted among CD4(+)CD8(+) thymocytes from DSS-treated mice. Also noteworthy was the 40% decline in cells of the erythrocytic lineages in the marrow of colitis mice, which undoubtedly contributed to the anemia observed in these mice. The peripheral blood reflected the marrow changes as demonstrated by a 2.6-fold increase in neutrophils, a 60% increase in monocytes, and a decline in the lymphocyte population. Thus, colitis changed the BM in profound ways that parallel the general outcomes of colitis including infiltration of the gut with monocytes and neutrophils, inflammation, and anemia. The data provide important understandings of the full impact of colitis that may lead to unique treatments and therapies.

Ultrasound Education in an Elective Course in the Undergraduate Medical Curriculum Improves Students' Subjective Learning and Clinical Rotation and Residency Preparedness.

Objectives: Studies have demonstrated that students are able to improve their understanding of anatomy, physical exam skills, and clinical knowledge when ultrasound is incorporated into their education. This study investigates whether students enrolled in an elective with significant use of ultrasound (ACS) in their preclinical years subjectively improved their preparedness for clinical rotations, residency, and learning in several subjects. Methods: This was a retrospective analysis of an anonymous online needs assessment survey sent to 384 third- and fourth-year medical students. Survey items included Likert scales analyzed with a student's t-test or Chi-Square analysis and yes/no questions analyzed with Chi square, as well as demographic information. Results: 201 students responded to the survey (52.3% response rate). ACS participants reported feeling more prepared for their clinical rotations than their non-ACS peers (3.5 vs. 2.33, p < 0.001 ) and felt more prepared for residency (3.12 vs 1.91, p < 0.001). ACS students also found ultrasound significantly more helpful in understanding the physical exam (69.3% vs. 53.2%, p = 0.02) and anatomy (94.7% vs. 81%, p = 0.012). Conclusions: Student respondents expressed a desire for more ultrasound education throughout medical school. Students who participated in ACS reported feeling more prepared for their clinical rotations and residency than their non-ACS peers and were more likely to feel that ultrasound improved their understanding of anatomy and the physical exam. This study adds to the literature suggesting that increasing the quantity of ultrasound in preclinical medical education may improve students' satisfaction, learning and clinical preparedness. Supplementary Information: The online version contains supplementary material available at 10.1007/s40670-023-01800-4.

Quality and Implementation of Diabetic Care at a Free Clinic.

INTRODUCTION: Although typically receiving little government funding, free clinics help ensure access to affordable quality health care to the medically underserved. Established in 2004, the authors' metropolitan Detroit Health Unit on Davison Avenue (HUDA) Clinic delivers primary care and specialized services to uninsured populations. The authors compared proportionate changes in A1c levels compared to prior national averages to evaluate the significance of care a free diabetes clinic can provide to uninsured populations. METHODS: Retrospective data from 2017-2019 were reviewed of adult patients who have been diagnosed with Type 2 Diabetes. From HUDA Clinic medical records, data were collected concerning patient demographics, insurance status, pregnancy, major comorbidities and several factors related to diabetes standards of care. RESULTS: There were a total of 2,231 patient visits to HUDA Clinic in 2019, of which 125 patients (5.6%) received care for their Type 2 diabetes. Forty (32%) clinic patients who had a visit in 2019 had an HbA1c <7.0 and 29 (23%) had an HbA1c > 9.5. This is comparable to the 2020 National Diabetes Statistics Report in which approximately 50% (n = 15.6 million) of Americans had an HbA1c < 7.0 and 14% (n = 5.1 million) had an HbA1c > 9.5. CONCLUSIONS: Huda Clinic's diabetes care percentages were quite comparable to state and national data and CDC parameters, although these comparative results need to be considered in terms of the authors' smaller sample size. These overall results indicate that health care providers can meet major recommended diabetic care at inner-city free clinics in metropolitan communities. Future provider and patient study studies regarding free clinic care patterns are clearly required to identify gaps in healthcare access and formulate and test specific strategies to improve diabetes-related outcomes.

Natural glucocorticoids induce expansion of all developmental stages of murine bone marrow granulocytes without inhibiting function.

Natural glucocorticoids (Gc) produced during stress have profound effects on the immune system. It is well known that Gc induce apoptosis in precursor T and B cells, markedly altering lymphopoiesis. However, it has been noted that marrow myeloid cells expanded both in proportion and absolute numbers in the mouse after Gc exposure. Mice were implanted with a corticosterone (CS) tablet that increased serum Gc and caused atrophied thymuses, both classic signs of activation of the stress axis. Blood neutrophil counts were elevated (4.8x), whereas lymphocyte counts declined. Flow cytometric analysis of the marrow revealed that the phenotypic distribution of the various major classes of cells was shifted by Gc exposure. As expected, marrow lymphocyte numbers declined >40% after 3 days of exposure to Gc. Conversely, in the myeloid compartment, both monocytes and granulocytes increased in number by >40%. Further, all granulocyte developmental stages showed large increases in both total number and percentage of cells. To investigate the functional capacity of mature granulocytes from Gc-treated mice, an improved granulocyte isolation method was developed. Gc exposure had little effect on the ability of granulocytes to produce superoxide or undergo chemotaxis or phagocytose bacteria. These results indicate that Gc treatment shifts bone marrow composition and provides evidence that granulocytes and their progenitors are selectively preserved under stressful conditions without losing function.

Theiler's virus persistence in the central nervous system of mice is associated with continuous viral replication and a difference in outcome of infection of infiltrating macrophages versus oligodendrocytes.

Theiler's murine encephalomyelitis virus (TMEV) infection of mice, in which persistent central nervous system (CNS) infection induces Th1 CD4+ T cell responses to both virus and myelin proteins, provides a relevant experimental animal model for MS. During persistence, >10(9) TMEV genome equivalents per spinal cord are detectable by real-time reverse transcription-polymerase chain reaction (RT-PCR). Because of the short half-life of TMEV (<1 day), continual viral replication is needed to sustain these very high TMEV copy numbers. An essential role for macrophages in TMEV persistence has been documented and, although limited by host anti-viral immune responses, TMEV nonetheless spreads during persistence to infect other cells, particularly oligodendrocytes, in which the infection is productive and lytic. Virus factors influencing persistence of TMEV are expression of the out-of-frame L* protein and use of sialic acid co-receptors.

Susceptibility of peritoneal macrophages to infection by Theiler's virus.

Theiler's murine encephalomyelitis virus (TMEV) strains fall into two groups: high-neurovirulence GDVII virus results in rapidly fatal encephalitis, while low-neurovirulence BeAn and DA viruses produce persistent central nervous system (CNS) infection and inflammatory demyelinating disease. Because macrophages (Mphis) are key components in BeAn virus-induced demyelinating disease, we examined the susceptibility of primary peritoneal macrophages (pMphis) to BeAn infection in vitro. Freshly isolated, thioglycollate-elicited pMphis were resistant to BeAn virus infection even at high multiplicity of infection. In contrast, after incubation of thioglycollate-elicited pMphis at 37 degrees C for 4 days before infection, approximately half of the cells expressed virus antigen(s) and contained nicked DNA indicative of apoptosis. However, BeAn virus RNA replication and virus yields were highly restricted. Interestingly, about one-third of the cells were apoptotic but negative for virus RNA and antigen(s). Tumor necrosis factor-alpha (TNF-alpha) and interferon-alpha (IFN-alpha) were elevated in BeAn-infected pMphi cultures suggesting that bystander killing may be responsible for the apoptosis seen in BeAn virus antigen-negative cells. These data show for the first time that pMphis are susceptible to BeAn virus infection, although the infection is highly restricted and most of these cells undergo BeAn-induced apoptosis.

Enhanced detection of Theiler's virus RNA copy equivalents in the mouse central nervous system by real-time RT-PCR.

Infection of mice by low-neurovirulence Theiler's murine encephalomyelitis virus (TMEV), such as BeAn and DA viruses, provides a relevant experimental animal model for multiple sclerosis (MS). As a step toward determining the kinetics of a persistent central nervous system (CNS) infection that leads to chronic demyelination, we adapted a rapid, accurate and highly specific real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection and quantitation of BeAn virus RNA copy equivalents in mouse tissues. The assay enabled detection of as few as 20-30 copies of BeAn virus RNA per microg of total RNA from infected mouse tissues and results for spinal cord revealed the same high levels of BeAn RNA as detected by Northern hybridization during the first 4 months of the persistent infection, but also was able to detect virus RNA copies as late as 1 year post-infection. Real-time RT-PCR analysis of BeAn virus RNA copy equivalents in different parts of the CNS, analyses not possible by Northern hybridization, revealed the following cline of virus persistence: spinal cord>brainstem/cerebellum>cerebrospinal fluid (CSF)>cerebral hemispheres. Systemic organs, including heart, intestine and mesenteric lymph nodes of infected mice, showed no evidence of viral persistence at 4 months post-infection.

Functional capacity of neutrophils from class III obese patients.

Class III obesity is associated with chronic inflammation and a variety of changes in immune function. Yet surprisingly little was known about the status of neutrophils that represent the first line of immune defense. The aim of this study was to assess key functions of neutrophils from class III obese patients, namely phagocytosis, superoxide production, chemotaxis, and response to endotoxin challenge, and compare their responses with lean controls. Thirty obese patients (BMI 48.8 ± 6.6 kg/m(2)) with comorbidities such as diabetes, hyperlipidemia, high blood pressure, etc. and nine lean (BMI between 20 and 25) subjects were enrolled in the study. Neutrophils from class III obese patients phagocytosed Escherichia coli (E. coli) at similar rates and with adequate numbers of bacteria taken up per cell compared with cells from lean subjects. Neutrophil production of superoxide, which is key to rapid killing of pathogens, showed modest diminution in the class III obese, which increased among patients with BMI >50. Chemotactic activity of neutrophils from class III obese patients was not altered. However, neutrophils from obese subjects showed an increased response to low-dose endotoxin, with concomitant reduced apoptosis and extension of their half-life compared with lean subjects, which suggests possible hyperresponsiveness of these neutrophils. Overall, neutrophil activity was not significantly altered by age, gender, diabetic status, or hyperlipidemia. Collectively, these results suggest that class III obese patients, even with comorbidities, have normal or nearly normal phagocytic, chemotactic, and superoxide generating capacity.

Peripheral, but not central nervous system, type I interferon expression in mice in response to intranasal vesicular stomatitis virus infection.

Type I interferon (IFN) is critical for resistance of mice to infection with vesicular stomatitis virus (VSV). Wild type (wt) VSV infection did not induce type I IFN production in vitro or in the central nervous system (CNS) of mice; however IFN-beta was detected in lungs, spleen, and serum within 24 h. The M protein mutant VSV, T1026R1 (also referred to as M51R), induced type I IFN production in vitro and in the CNS, with poor expression in spleens. In addition, VSV T1026R1 was not pathogenic to mice after intranasal infection, illustrating the importance of IFN in controlling VSV replication in the CNS. Experiments with chemical sympathectomy, sRAGE, and neutralizing antibody to VSV were performed to investigate the mechanism(s) utilized for induction of peripheral IFN; neither sRAGE infusion nor chemical sympathectomy had an effect on peripheral IFN production. In contrast, administration of neutralizing antibody (Ab) readily blocked the response. Infectious VSV was transiently present in lungs and spleens at 24 h post infection. The results are consistent with VSV traffic from the olfactory neuroepithelium to peripheral lymphoid organs hematogenously or via lymphatic circulation. These results suggest that VSV replicates to high titers in the brains of mice because of the lack of IFN production in the CNS after intranasal VSV infection. In contrast, replication of VSV in peripheral organs is controlled by the production of large amounts of IFN.

VSV replication in neurons is inhibited by type I IFN at multiple stages of infection.

Vesicular stomatitis virus (VSV) is a rhabdovirus which causes acute encephalitis in mice after intranasal infection. Because type I interferon (IFN) has been shown to be a potent inhibitor of VSV, we investigated the role of type I IFN in viral replication in neurons in culture. Pre-treatment of NB41A3 neuroblastoma cells or primary neuron cultures with IFN-beta or IFN-alpha strongly inhibits virus replication, with 1000-fold inhibition of infectious virus release occurring at 7 h post-infection, and maximum inhibition of 14,000-fold occurring at 14 h. Type I IFN inhibited both viral protein and RNA synthesis, but not enough to account for the inhibition of infectious virus yield. The influenza virus protein NS1 binds dsRNA and antagonizes induction of PKR activity, an IFN-inducible antiviral protein which phosphorylates and inactivates the elongation factor eIF-2alpha, resulting in cessation of translation. In NS1-expressing neuroblastoma cells, VSV replication was inhibited by IFN-beta as well as in control NB41A3 cells, and eIF-2alpha phosphorylation was blocked, suggesting that PKR activity was not involved in inhibition of viral protein synthesis. Similarly, inhibition of VSV by IFN-beta was not affected by addition of inhibitors of nitric oxide synthase, indicating that IFN-beta activity is not mediated by nitric oxide or superoxide. This contrasts with the essential role of NOS-1 in inhibition of VSV replication when neurons are treated with IFN-gamma. Analysis of cell culture supernatants revealed suppression of release of VSV particles from both NB41A3 cells and primary neurons treated with IFN. The inhibition of virion release closely matched the overall suppression of infectious VSV particle release, suggesting that type I IFN plays a role in inhibition of VSV assembly.

Selection and characterization of a BHK-21 cell line resistant to infection by Theiler's murine encephalomyelitis virus due to a block in virus attachment and entry.

A clonal population of BHK-21 cells resistant to infection with the low-neurovirulence BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) was derived after four cycles of infection and characterized. These cells were resistant to both low- and high-neurovirulence TMEV strains due to a block in virus attachment and entry and not in virus replication, since transfection of these cells with TMEV RNA to bypass the entry step(s) induced virus replication and assembly. The resistance to infection was stable for more than a year, suggesting that it is a heritable property arising from a mutation in the susceptible parent BHK-21 population. This cell line is being used to identify a receptor for TMEV.

Transition from acute to persistent Theiler's virus infection requires active viral replication that drives proinflammatory cytokine expression and chronic demyelinating disease.

The dynamics of Theiler's murine encephalomyelitis virus (TMEV) RNA replication in the central nervous systems of susceptible and resistant strains of mice were examined by quantitative real-time reverse transcription-PCR and were found to correlate with host immune responses. During the acute phase of infection in both susceptible and resistant mice, levels of viral replication were high in the brain and brain stem, while levels of viral genome equivalents were 10- to 100-fold lower in the spinal cord. In the brain, viral RNA replication decreased after a peak at 5 days postinfection (p.i.), in parallel with the appearance of virus-specific antibody responses; however, by 15 days p.i., viral RNA levels began to increase in the spinal cords of susceptible mice. During the transition to and the persistent phase of infection, the numbers of viral genome equivalents in the spinal cord varied substantially for individual mice, but high levels were consistently associated with high levels of proinflammatory Th1 cytokine and chemokine mRNAs. Moreover, a large number of viral genome equivalents and high proinflammatory cytokine mRNA levels in spinal cords were only observed for susceptible SJL/J mice who developed demyelinating disease. These results suggest that TMEV persistence requires active viral replication beginning about day 11 p.i. and that active viral replication with high viral genome loads leads to increased levels of Th1 cytokines that drive disease progression in infected mice.