Detection of a Human Adenovirus Outbreak, Including Some Critical Infections, Using Multipathogen Testing at a Large University, September 2022-January 2023.

Background: Human adenoviruses (HAdVs) can cause outbreaks of flu-like illness in university settings. Most infections in healthy young adults are mild; severe illnesses rarely occur. In Fall 2022, an adenovirus outbreak was identified in university students. Methods: HAdV cases were defined as university students 17-26 years old who presented to the University Health Service or nearby emergency department with flu-like symptoms (eg, fever, cough, headache, myalgia, nausea) and had confirmed adenovirus infections by polymerase chain reaction (PCR). Demographic and clinical characteristics were abstracted from electronic medical records; clinical severity was categorized as mild, moderate, severe, or critical. We performed contact investigations among critical cases. A subset of specimens was sequenced to confirm the HAdV type. Results: From 28 September 2022 to 30 January 2023, 90 PCR-confirmed cases were identified (51% female; mean age, 19.6 years). Most cases (88.9%) had mild illness. Seven cases required hospitalization, including 2 critical cases that required intensive care. Contact investigation identified 44 close contacts; 6 (14%) were confirmed HAdV cases and 8 (18%) reported symptoms but never sought care. All typed HAdV-positive specimens (n = 36) were type 4. Conclusions: While most students with confirmed HAdV had mild illness, 7 otherwise healthy students had severe or critical illness. Between the relatively high number of hospitalizations and proportion of close contacts with symptoms who did not seek care, the true number of HAdV cases was likely higher. Our findings illustrate the need to consider a wide range of pathogens, even when other viruses are known to be circulating.

Regulation of amyloid-ß levels by matrix metalloproteinase-2/9 (MMP2/9) in the media of lung cancer cells.

In this study, we set out to identify regulators of intact amyloid-ß40/42 (Aß) levels in A549 (p53 wild-type) and H1299 (p53-null) lung cancer cell media. Higher Aß levels were detected in the media of A549 than H1299 cells without or with treatment with 4-methylumbelliferone (4-MU) and/or the anti-CD44 antibody (5F12). Using inhibitors, we found that PI3K, AKT, and NFκB are likely involved in regulating Aß levels in the media. However, increased Aß levels that more closely resembled those found upon 4-MU co-treatment resulted from MMP2/9 inhibition, suggesting that MMP2/9 maybe the main contributors to regulation of Aß levels in the media. Differences in Aß levels might be accounted for, in part, by p53 since blocking p53 function in A549 cells resulted in decreased Aß levels, increased MMP2/9 levels, increased PI3K/AKT activities and the phospho/total NFκB ratio. Using siRNA targeted against MMP2 or MMP9, we found increased Aß levels in the media, however, MMP2 knockdown led to Aß levels closely mimicking those detected by co-treatment with 4-MU. Cell viability or apoptosis upon treatment with either MMP2 or MMP9 siRNA along with Aß immunodepletion, showed that MMP2 is the predominant regulator of the cytotoxic effects induced by Aß in lung cancer cells.

Interaction of amyloid beta with humanin and acetylcholinesterase is modulated by ATP.

Humanin (HN) is known to bind amyloid beta (Aß)-inducing cytoprotective effects, while binding of acetylcholinesterase (AChE) to Aß increases its aggregation and cytotoxicity. Previously, we showed that binding of HN to Aß blocks aggregation induced by AChE and that HN decreases but does not abolish Aß-AChE interactions in A549 cell media. Here, we set out to shed light on factors that modulate the interactions of Aß with HN and AChE. We found that binding of either HN or AChE to Aß is not affected by heparan sulfate, while ATP, thought to reduce misfolding of Aß, weakened interactions between AChE and Aß but strengthened those between Aß and HN. Using media from either A549 or H1299 lung cancer cells, we observed that more HN was bound to Aß upon addition of ATP, while levels of AChE in a complex with Aß were decreased by ATP addition to A549 cell media. Exogenous addition of ATP to either A549 or H1299 cell media increased interactions of endogenous HN with Aß to a comparable extent despite differences in AChE expression in the two cell lines, and this was correlated with decreased binding of exogenously added HN to Aß. Treatment with exogenous ATP had no effect on cell viability under all conditions examined. Exogenously added ATP did not affect viability of cells treated with AChE-immunodepleted media, and there was no apparent protection against the cytotoxicity resulting from immunodepletion of HN. Moreover, exogenously added ATP had no effect on the relative abundance of oligomer versus total Aß in either cell line.

Biochemical determinants of the IGFBP-3-hyaluronan interaction.

IGFBP-3, the most abundant IGFBP and the main carrier of insulin-like growth factor I (IGF-I) in the circulation, can bind IGF-1 with high affinity, which attenuates IGF/IGF-IR interactions, thereby resulting in antiproliferative effects. The C-terminal domain of insulin-like growth factor-binding protein-3 (IGFBP-3) is known to contain an 18-basic amino acid motif capable of interacting with either humanin (HN) or hyaluronan (HA). We previously showed that the 18-amino acid IGFBP-3 peptide is capable of binding either HA or HN with comparable affinities to the full-length IGFBP-3 protein and that IGFBP-3 can compete with the HA receptor, CD44, for binding HA. Blocking the interaction between HA and CD44 reduced viability of A549 human lung cancer cells. In this study, we set out to better characterize IGFBP-3-HA interactions. We show that both stereochemistry and amino acid identity are important determinants of the interaction between the IGFBP-3 peptide and HA and for the peptide's ability to exert its cytotoxic effects. Binding of IGFBP-3 to either HA or HN was unaffected by glycosylation or reduction of IGFBP-3, suggesting that the basic 18-amino acid residue sequence of IGFBP-3 remains accessible for interaction with either HN or HA upon glycosylation or reduction of the full-length protein. Removing N-linked oligosaccharides from CD44 increased its ability to compete with IGFBP-3 for binding HA, while reduction of CD44 rendered the protein relatively ineffective at blocking IGFBP-3-HA interactions. We conclude that both deglycosylation and disulfide bond formation are important for CD44 to compete with IGFBP-3 for binding HA.

Humanin Blocks the Aggregation of Amyloid-ß Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3.

It is known that the humanin (HN) peptide binding to amyloid-ß (Aß) protects against its cytotoxic effects, while acetylcholinesterase (AChE) binding to Aß increases its aggregation and cytotoxicity. HN is also known to bind the insulin-like growth factor binding protein-3 (IGFBP-3). Here, we examined the regulation of Aß conformations by HN, AChE, and IGFBP-3 both in vitro and in the conditioned media from A549 and H1299 lung cancer cells. Our in vitro results showed the following: IGFBP-3 binds HN and blocks it from binding Aß in the absence or presence of AChE; HN and AChE can simultaneously bind Aß but not when in the presence of IGFBP-3; HN is unable to reduce the aggregation of Aß in the presence of IGFBP-3; and HN abolishes the aggregation of Aß induced by the addition of AChE in the absence of IGFBP-3. In the media, AChE and HN can simultaneously bind Aß. While both AChE and HN are detected when using 6E10 Aß antibodies, only AChE is detected when using the Aß 17-24 antibody 4G8, the anti-oligomer A11, and the anti-amyloid fibril LOC antibodies. No signal was observed for IGFBP-3 with any of the anti-amyloid antibodies used. Exogenously added IGFBP-3 reduced the amount of HN found in a complex when using 6E10 antibodies and correlated with a concomitant increase in the amyloid oligomers. Immunodepletion of HN from the media of the A549 and H1299 cells increased the relative abundance of the oligomer vs the total amount of Aß, the A11-positive prefibrillar oligomers, and to a lesser extent the LOC-positive fibrillar oligomers, and was also correlated with diminished cell viability and increased apoptosis.

IGFBP-3 Blocks Hyaluronan-CD44 Signaling, Leading to Increased Acetylcholinesterase Levels in A549 Cell Media and Apoptosis in a p53-Dependent Manner.

Insulin-like growth factor binding protein-3 (IGFBP-3) belongs to a family of six IGF binding proteins. We previously found that IGFBP-3 exerts its cytotoxic effects on A549 (p53 wild-type) cell survival through a mechanism that depends on hyaluronan-CD44 interactions. To shed light on the mechanism employed, we used CD44-negative normal human lung cells (HFL1), A549, and H1299 (p53-null) lung cancer cells. A synthetic IGFBP-3 peptide (215-KKGFYKKKQCRPSKGRKR-232) but not the mutant (K228AR230A), was able to bind hyaluronan more efficiently than the analogous sequences from the other IGFBPs. In a manner comparable to that of the IGFBP-3 protein, the peptide blocked hyaluronan-CD44 signaling, and more effectively inhibited viability of A549 cells than viability of either H1299 or HFL1 cell lines. Treatment with the IGFBP-3 protein or its peptide resulted in increased acetylcholinesterase concentration and activity in the A549 cell media but not in the media of either HFL1 or H1299, an effect that correlated with increased apoptosis and decreased cell viability. These effects were diminished upon the same treatment of A549 cells transfected with either p53 siRNA or acetylcholinesterase siRNA. Taken together, our results show that IGFBP-3 or its peptide blocks hyaluronan-CD44 signaling via a mechanism that depends on both p53 and acetylcholinesterase.

Using Small Peptide Segments of Amyloid-ß and Humanin to Examine their Physical Interactions.

BACKGROUND: Amyloid fibrils in Alzheimer's disease are composed of amyloid-ß (Aß) peptides of variant lengths. Humanin (HN), a 24 amino acid residue neuroprotective peptide, is known to interact with the predominant Aß isoform in the brain, Aß (1-40). METHODS: Here, we constructed smaller segments of Aß and HN and identified residues in HN important for both HN-HN and HN-Aß interactions. Peptides corresponding to amino acid residues 5- 15 of HN, HN (5-15), HN (5-15, L11S), where Leu11 was replaced with Ser, and residues 17-28 of Aß, Aß (17-28), were synthesized and tested for their ability to block formation of the complex between HN and Aß (1-40). RESULTS: Co-immunoprecipitation and binding kinetics showed that HN (5-15) was more efficient at blocking the complex between HN and Aß (1-40) than either HN (5-15, L11S) or Aß (17-28). Binding kinetics of these smaller peptides with either full-length HN or Aß (1-40) showed that HN (5- 15) was able to bind either Aß (1-40) or HN more efficiently than HN (5-15, L11S) or Aß (17-28). Compared to full-length HN, however, HN (5-15) bound Aß (1-40) with a weaker affinity suggesting that while HN (5-15) binds Aß, other residues in the full length HN peptide are necessary for maximum interactions. CONCLUSION: L11 was more important for interactions with Aß (1-40) than with HN. Aß (17-28) was relatively ineffective at binding to either Aß (1-40) or HN. Moreover, HN, and the smaller HN (5-15), HN (5-15 L11S), and Aß (17-28) peptides, had different effects on regulating Aß (1-40) aggregation kinetics.

Interaction of Insulin-Like Growth Factor-Binding Protein 3 With Hyaluronan and Its Regulation by Humanin and CD44.

Insulin-like growth factor-binding protein-3 (IGFBP-3) belongs to a family of IGF-binding proteins. Humanin is a peptide known to bind residues 215-232 of mature IGFBP-3 in the C-terminal region of the protein. This region of IGFBP-3 was shown earlier to bind certain glycosaminoglycans including hyaluronan (HA). Here, we characterized the binding affinities of the IGFBP-3 protein and peptide (215-KKGFYKKKQCRPSKGRKR-232) to HA and to humanin and found that HA binds with a weaker affinity to this region than does humanin. Either HA or humanin could bind to this IGFBP-3 segment, but not simultaneously. The HA receptor, CD44, blocked HA binding to IGFBP-3 but had no effect on binding of humanin to either IGFBP-3 or its peptide. Upon incubation of HA with CD44 and either IGFBP-3 protein or peptide, humanin was effective at binding and sequestering IGFBP-3 or peptide, thereby enabling access of CD44 to HA. We show that IGFBP-3 and humanin in the medium of A549 lung cancer cells can immunoprecipitate in a complex. However, the fraction of IGFBP-3 in the medium that is able to bind HA was not complexed with humanin suggesting that HA binding to the 215-232 segment renders it inaccessible for binding to humanin. Moreover, while the cytotoxic effects of IGFBP-3 on cell viability were reversed by humanin, blocking HA-CD44 interaction with an anti-CD44 antibody in combination with IGFBP-3 did not have an additive negative effect on cell viability suggesting that IGFBP-3 exerts its cytotoxic effects on cell survival through a mechanism that depends on HA-CD44 interactions.