Hepatitis A Virus Infections Among Men Who Have Sex with Men - Eight U.S. States, 2017-2018.

During 1995-2011, the overall incidence of hepatitis A decreased by 95% in the United States from 12 cases per 100,000 population during 1995 to 0.4 cases per 100,000 population during 2011, and then plateaued during 2012─2015. The incidence increased by 294% during 2016-2018 compared with the incidence during 2013-2015, with most cases occurring among populations at high risk for hepatitis A infection, including persons who use illicit drugs (injection and noninjection), persons who experience homelessness, and men who have sex with men (MSM) (1-3). Previous outbreaks among persons who use illicit drugs and MSM led to recommendations issued in 1996 by the Advisory Committee on Immunization Practices (ACIP) for routine hepatitis A vaccination of persons in these populations (4). Despite these long-standing recommendations, vaccination coverage rates among MSM remain low (5). In 2017, the New York City Department of Health and Mental Hygiene contacted CDC after public health officials noted an increase in hepatitis A infections among MSM. Laboratory testing* of clinical specimens identified strains of the hepatitis A virus (HAV) that subsequently matched strains recovered from MSM in other states. During January 1, 2017-October 31, 2018, CDC received reports of 260 cases of hepatitis A among MSM from health departments in eight states, a substantial increase from the 16 cases reported from all 50 states during 2013-2015. Forty-eight percent (124 of 258) of MSM patients were hospitalized for a median of 3 days. No deaths were reported. In response to these cases, CDC supported state and local health departments with public health intervention efforts to decrease HAV transmission among MSM populations. These efforts included organizing multistate calls among health departments to share information, providing guidance on developing targeted outreach and managing supplies for vaccine campaigns, and conducting laboratory testing of clinical specimens. Targeted outreach for MSM to increase awareness about hepatitis A infection and improve access to vaccination services, such as providing convenient locations for vaccination, are needed to prevent outbreaks among MSM.

Neonatal seizures are associated with redistribution and loss of GABAA α-subunits in the hypoxic-ischaemic pig.

Seizures are a common manifestation of hypoxic-ischaemic brain injury in the neonate. In status epilepticus models alterations to GABAA R subunit expression have been suggested to contribute to (i) abnormal development of the GABAergic system, (ii) why seizures become self-sustaining and (iii) the development of pharmacoresistance. Detailed investigation of GABAA R subunit protein expression after neonatal hypoxia-ischaemia (HI) is currently insufficient. Using our pig model of HI and subsequent spontaneous neonatal seizures, we investigated changes in protein expression of the three predominant α-subunits of the GABAA R; α1 , α2 and α3 . Anaesthetized, ventilated newborn pigs (< 24 h old) were subjected to 30 min HI and subsequently recovered to 24 or 72 h. Amplitude-integrated electroencephalography was used to monitor brain activity and identify seizure activity. Brain tissue was collected post-mortem and GABAA R α-subunit protein expression was analysed using western blot and immunohistochemistry. GABAA R α1 and α3 protein expression was significantly reduced in animals that developed seizures after HI; HI animals that did not develop seizures did not exhibit the same reductions. Immunohistochemistry revealed decreased α1 and α3 expression, and α1 redistribution from the cell membrane to the cytosol, in the hippocampus of seizure animals. Multivariate analyses, controlling for HI severity and neuronal injury, revealed that seizures were independently associated with significant GABAA R α3 reduction. This is the first study to show loss and redistribution of GABAA R α-subunits in a neonatal brain experiencing seizures. Our findings are similar to those reported in models of SE and in chronic epilepsy.

Phosphorylation of GFAP is associated with injury in the neonatal pig hypoxic-ischemic brain.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in the astrocyte cytoskeleton that plays an important role in the structure and function of the cell. GFAP can be phosphorylated at six serine (Ser) or threonine (Thr) residues but little is known about the role of GFAP phosphorylation in physiological and pathophysiological states. We have generated antibodies against two phosphorylated GFAP (pGFAP) proteins: p8GFAP, where GFAP is phosphorylated at Ser-8 and p13GFAP, where GFAP is phosphorylated at Ser-13. We examined p8GFAP and p13GFAP expression in the control neonatal pig brain and at 24 and 72 h after an hypoxic-ischemic (HI) insult. Immunohistochemistry demonstrated pGFAP expression in astrocytes with an atypical cytoskeletal morphology, even in control brains. Semi-quantitative western blotting revealed that p8GFAP expression was significantly increased at 24 h post-insult in HI animals with seizures in frontal, parietal, temporal and occipital cortices. At 72 h post-insult, p8GFAP and p13GFAP expression were significantly increased in HI animals with seizures in brain regions that are vulnerable to cellular damage (cortex and basal ganglia), but no changes were observed in brain regions that are relatively spared following an HI insult (brain stem and cerebellum). Increased pGFAP expression was associated with poor neurological outcomes such as abnormal encephalography and neurobehaviour, and increased histological brain damage. Phosphorylation of GFAP may play an important role in astrocyte remodelling during development and disease and could potentially contribute to the plasticity of the central nervous system.

Perceptions of tuberculosis among immigrants and refugees at an adult education center: a community-based participatory research approach.

English as a Second Language programs serve large foreign-born populations in the US with elevated risks of tuberculosis (TB), yet little is known about TB perceptions in these settings. Using a community-based participatory research approach, we elicited perceptions about TB among immigrant and refugee learners and staff at a diverse adult education center. Community partners were trained in focus groups moderation. Ten focus groups were conducted with 83 learners and staff. Multi-level, team-based qualitative analysis was conducted to develop themes that informed a model of TB perceptions among participants. Multiple challenges with TB control and prevention were identified. There were a variety of misperceptions about transmission of TB, and a lack of knowledge about latent TB. Feelings and perceptions related to TB included secrecy, shame, fear, and isolation. Barriers to TB testing include low awareness, lack of knowledge about latent TB, and the practical considerations of transportation, cost, and work schedule conflicts. Barriers to medication use include suspicion of generic medications and perceived side effects. We posit adult education centers with large immigrant and refugee populations as excellent venues for TB prevention, and propose several recommendations for conducting these programs. Content should dispel the most compelling misperceptions about TB transmission while clarifying the difference between active and latent disease. Learners should be educated about TB in the US and that it is curable. Finally, TB programs that include learners and staff in their design and implementation provide greater opportunity for overcoming previously unrecognized barriers.

Morphological changes in white matter astrocytes in response to hypoxia/ischemia in the neonatal pig.

White matter damage is a significant problem in the human pre-term baby. Damage to white matter is usually associated with injury or insults to babies born prematurely, typically before 32weeks' gestation, however there is increasing evidence of both grey and white matter damage occurring after 32weeks' gestation. Astrocytes play a vital role in white matter, regulating molecules such as glutamate in the extracellular space and preventing excitotoxic damage to neighbouring oligodendrocytes and axons. We have previously described dramatic changes in grey matter astrocytes in response to a hypoxic/ischemic (H/I) insult around the time of birth. In this study, we have used GFAP immunohistochemistry and Golgi-Kopsch staining to examine the morphology of white matter astrocytes in control neonatal pig brains, and in the brains of animals exposed to the same (perinatal) H/I insult. We demonstrate that the areal percentage of the section occupied by GFAP-immunoreactive processes and cell bodies is significantly decreased (by 46%, P<0.0001) in subcortical white matter from H/I brains. This loss of GFAP was accompanied by alterations in astrocyte morphology and an overall decrease in the size (field of section occupied by an individual astrocyte) of white matter astrocytes from 649microm(2) to 426microm(2), as revealed by Golgi-Kopsch staining and image analysis. These data suggest that astrocytes may contribute to the pathology of white matter damage following an H/I insult around the time of birth, and suggest that astrocytes may offer a novel target for therapies to improve outcomes after H/I.

Structural remodeling of gray matter astrocytes in the neonatal pig brain after hypoxia/ischemia.

Astrocytes play a vital role in the brain; their structural integrity and sustained function are essential for neuronal viability, especially after injury or insult. In this study, we have examined the response of astrocytes to hypoxia/ischemia (H/I), employing multiple methods (immunohistochemistry, iontophoretic cell injection, Golgi-Kopsch staining, and D-aspartate uptake) in a neonatal pig model of H/I. We have identified morphological changes in cortical gray matter astrocytes in response to H/I. Initial astrocytic changes were evident as early as 8 h post-insult, before histological evidence for neuronal damage. By 72 h post-insult, astrocytes exhibited significantly fewer processes that were shorter, thicker, and had abnormal terminal swellings, compared with astrocytes from control brains that exhibited a complex structure with multiple fine branching processes. Quantification and image analysis of astrocytes at 72 h post-insult revealed significant decreases in the average astrocyte size, from 686 microm(2) in controls to 401 microm(2) in H/I brains. Sholl analysis revealed a significant decrease (>60%) in the complexity of astrocyte branching between 5 and 20 microm from the cell body. D-Aspartate uptake studies revealed that the H/I insult resulted in impaired astrocyte function, with significantly reduced clearance of the glutamate analog, D-aspartate. These results suggest that astrocytes may be involved in the pathophysiological events of H/I brain damage at a far earlier time point than first thought. Developing therapies that prevent or reverse these astrocytic changes may potentially improve neuronal survival and thus might be a useful strategy to minimize brain damage after an H/I insult.

Genetic determination of the effect of post-translational modification on the innate immune response to the 19 kDa lipoprotein of Mycobacterium tuberculosis.

BACKGROUND: The 19 kDa lipoprotein of Mycobacterium tuberculosis (MTB) is an important target of the innate immune response. To investigate the effect of post-translation modification of this protein on innate recognition in the context of the whole bacillus, we derived a recombinant M. tuberculosis H37Rv that lacked the 19 kDa gene (Delta19) and complemented this strain by reintroduction of the 19 kDa gene into the chromosome as a single copy to produce Delta19::19. We also reintroduced the 19 kDa gene in two modified forms that lacked motifs for acylation (Delta19::19NA) and O-glycosylation (Delta19::19NOG). RESULTS: Both acylation and O-glycosylation were necessary for the protein to remain within the cell. IL-1 Beta secretion from human monocytes was significantly reduced by deletion of the 19 kDa gene (p < 0.02). Complementation by the wild type, but not the mutagenised gene reversed this phenotype. The effect of deletion and complementation on IL-12p40 and TNF secretion was less marked with no statistically significant differences between strains. Although deletion of the 19 kDa reduced apoptosis, an effect that could also only be reversed by complementation with the wild type gene, the results were variable between donors and did not achieve statistical significance. CONCLUSION: These results confirm in the context of the whole bacillus an important role for post-translational modification of the 19 kDa on both the cellular location and immune response to this protein.

MAP2 provides reliable early assessment of neural injury in the newborn piglet model of birth asphyxia.

Reduction in microtubule-associated-protein-2 (MAP2) immunoreactivity is a sensitive and quantifiable early marker of neural injury in rats. This study assessed the reliability of MAP2 as an early marker of neural injury following hypoxia/ischaemia in neonatal piglets, and compared the effects of perfusion and immersion fixation on MAP2 immunoreactivity. Hypoxia was induced in newborn piglets (n=23) by reducing the FiO2 to 4% for 0, 25, 35 or 50 min. Six hours after the end of hypoxia piglets were killed, and the brain removed and immunolabelled for MAP2. Significant reductions in MAP2 immunoreactivity were seen in cortex, hippocampus, basal ganglia and thalamus. Reductions correlated with duration of hypoxia, pH at the end of hypoxia, cerebral function monitor amplitude and cerebral impedance 6h after hypoxia, and with early histological evidence of ischaemic changes. Regions with reduced immunoreactivity correlated with areas where damage is present in later histological examination in this model. Immersion fixation with postmortem delays up to 30 min did not affect MAP2 immunoreactivity compared to perfusion-fixed tissue. Results indicate that MAP2 immunoreactivity 6h after hypoxia/ischaemia is a reliable marker of neural injury in the neonatal piglet.

Cytoskeletal anchoring of GLAST determines susceptibility to brain damage: an identified role for GFAP.

Glial fibrillary acidic protein (GFAP) is an enigmatic protein; it currently has no unambiguously defined role. It is expressed in the cytoskeleton of astrocytes in the mammalian brain. We have used co-immunoprecipitation to identify in vivo binding partners for GFAP in the rat and pig brain. We demonstrate interactions between GFAP, the glutamate transporter GLAST, the PDZ-binding protein NHERF1, and ezrin. These interactions are physiologically relevant; we demonstrate in vitro that transport of D-aspartate (a glutamate analogue) is significantly increased in the presence of GFAP and NHERF1. Moreover, we demonstrate in vivo that expression of GFAP is essential in retaining GLAST in the plasma membranes of astrocytes after an hypoxic insult. These data indicate that the cytoskeleton of the astrocyte plays an important role in protecting the brain against glutamate-mediated excitotoxicity.

G-protein control of the ribosome-associated stress response protein SpoT.

The bacterial response to stress is controlled by two proteins, RelA and SpoT. RelA generates the alarmone (p)ppGpp under amino acid starvation, whereas SpoT is responsible for (p)ppGpp hydrolysis and for synthesis of (p)ppGpp under a variety of cellular stress conditions. It is widely accepted that RelA is associated with translating ribosomes. The cellular location of SpoT, however, has been controversial. SpoT physically interacts with the ribosome-associated GTPase CgtA, and we show here that, under an optimized salt condition, SpoT is also associated with a pre-50S particle. Analysis of spoT and cgtA mutants and strains overexpressing CgtA suggests that the ribosome associations of SpoT and CgtA are mutually independent. The steady-state level of (p)ppGpp is increased in a cgtA mutant, but the accumulation of (p)ppGpp during amino acid starvation is not affected, providing strong evidence that CgtA regulates the (p)ppGpp level during exponential growth but not during the stringent response. We show that CgtA is not associated with pre-50S particles during amino acid starvation, indicating that under these conditions in which (p)ppGpp accumulates, CgtA is not bound either to the pre-50S particle or to SpoT. We propose that, in addition to its role as a 50S assembly factor, CgtA promotes SpoT (p)ppGpp degradation activity on the ribosome and that the loss of CgtA from the ribosome is necessary for maximal (p)ppGpp accumulation under stress conditions. Intriguingly, we found that in the absence of spoT and relA, cgtA is still an essential gene in Escherichia coli.

In vivo functional characterization of the Saccharomyces cerevisiae 60S biogenesis GTPase Nog1.

The Saccharomyces cerevisiae Nog1 GTPase is critical for assembly of the large ribosomal subunit. Mutations in conserved residues in the GTP-binding pocket cause defects in cell growth and 60S ribosome assembly but mutant proteins retain their ability to associate with the pre-60S. Association of Nog1 with the pre-60S is independent of guanine nucleotide added to cell extracts. Thus, it appears that nucleotide occupancy does not substantially affect Nog1 association with pre-60S particles. Somewhat surprisingly, neither of the conserved threonines in the G2 motif of the GTPase domain is essential for Nog1 function. Neither the steady-state rRNA levels nor the protein composition (as determined by isobaric labeling and identification by mass spectrometry of peptides) of the pre-60S particles in the nog1P176V mutant are grossly perturbed, although levels of four proteins (Nog1, Nop2, Nop15, and Tif6) are modestly reduced in pre-60S particles isolated from the mutant. Deletion analysis revealed that the C-terminal 168 amino acids are not required for function; however, the N-terminal 126 amino acids are required. Optimal association with pre-60S particles requires sequences between amino acids 347-456. Several conserved charge-to-alanine substitutions outside the GTPase domain display modest growth phenotypes indicating that these residues are not critical for function.

Cooperative and critical roles for both G domains in the GTPase activity and cellular function of ribosome-associated Escherichia coli EngA.

To probe the cellular phenotype and biochemical function associated with the G domains of Escherichia coli EngA (YfgK, Der), mutations were created in the phosphate binding loop of each. Neither an S16A nor an S217A variant of G domain 1 or 2, respectively, was able to support growth of an engA conditional null. Polysome profiles of EngA-depleted cells were significantly altered, and His(6)-EngA was found to cofractionate with the 50S ribosomal subunit. The variants were unable to complement the abnormal polysome profile and were furthermore significantly impacted with respect to in vitro GTPase activity. Together, these observations suggest that the G domains have a cooperative function in ribosome stability and/or biogenesis.

Effect of deletion or overexpression of the 19-kilodalton lipoprotein Rv3763 on the innate response to Mycobacterium tuberculosis.

The 19-kDa lipoprotein of Mycobacterium tuberculosis is an important target of the innate immune response. To investigate the immune biology of this antigen in the context of the whole bacillus, we derived a recombinant M. tuberculosis H37Rv that lacked the 19-kDa-lipoprotein gene (Delta19) and complemented this strain by reintroduction of the 19-kDa-lipoprotein gene on a multicopy vector to produce Delta19::pSMT181. The Delta19 strain multiplied less well than Delta19::pSMT181 in human monocyte-derived macrophages (MDM) (P = 0.039). Surface expression of major histocompatibility complex class II molecules was reduced in phagocytes infected with M. tuberculosis; this effect was not seen in cells infected with Delta19. Delta19 induced lower interleukin 1beta (IL-1beta) secretion from monocytes and MDM. Overexpression of the 19-kDa protein increased IL-1beta, IL-12p40, and tumor necrosis factor alpha secretion irrespective of phagocyte maturity. These data support reports that the 19-kDa lipoprotein has pleiotropic effects on the interaction of M. tuberculosis with phagocytes. However, this analysis indicates that in the context of the whole bacillus, the 19-kDa lipoprotein is only one of a number of molecules that mediate the innate response to M. tuberculosis.