Gene therapy vector-related myocarditis.

Gene therapy is a technique to correct genetic abnormalities, through introduction of a functional gene or through direct genome editing. Adeno-associated virus (AAV)-mediated gene replacement shows promise for targeted therapies in treatment of inherited cardiomyopathies and is the most used approach in clinical trials. However, immune responses from the host to the virus and gene product pose delivery and safety challenges. This review explores the immunological reactions to AAV-based gene therapy, their potential toxic effects, with a focus on myocarditis, and future directions for gene therapy.

2021 Annual Report of the Kansas Poison Control Center at The University of Kansas Health System.

Introduction: This is the 2021 Annual Report of the Kansas Poison Control Center (KSPCC) at The University of Kansas Health System. The KSPCC serves the state of Kansas 24-hours a day, 365 days a year with certified specialists in poison information and clinical and medical toxicologists. Methods: Encounters reported to the KSPCC from January 1, 2021 through December 31, 2021 were analyzed. Data recorded includes caller demographics, exposure substance, nature and route of exposure, interventions, medical outcome, disposition, and location of care. Results: The KSPCC logged 18,253 total encounters in 2021, including calls from every county in Kansas. A majority of human exposure cases (53.6%) were female. Approximately 59.8% were pediatric exposures (defined as 19 years of age or less). Most encounters occurred at a residence (91.7%) and most were managed there (70.5%). Unintentional exposures were the most common reason for exposures (70.5%). The most common reported substance in pediatric encounters was household cleaning products (n = 815) and cosmetics/personal care products (n = 735). For adult encounters, analgesics (n = 1,241) and sedative/ hypnotics/antipsychotics (n = 1,013) were the most frequently reported. Medical outcomes were 26.0% no effect, 22.4% minor effect, 10.7% moderate effect, and 2.7% major effects. There were 22 deaths. Conclusions: The 2021 KSPCC annual report demonstrated that cases were received from the entire state of Kansas. Pediatric exposures remained most common but cases with serious outcomes continued to increase. This report supported the continued value of the KSPCC to both public and health care providers in the state of Kansas.

Adapting Patient and Public Involvement processes in response to the Covid-19 pandemic.

BACKGROUND: The COVID-19 pandemic brought rapid and major changes to research, and those wishing to carry out Patient and Public Involvement (PPI) activities faced challenges, such as restrictions on movement and contact, illness, bereavement and risks to potential participants. Some researchers moved PPI to online settings during this time but remote consultations raise, as well as address, a number of challenges. It is important to learn from PPI undertaken in this period as face-to-face consultation may no longer be the dominant method for PPI. METHODS: UK stay-at-home measures announced in March 2020 necessitated immediate revisions to the intended face-to-face methods of PPI consultation for the ESORT Study, which evaluated emergency surgery for patients with common acute conditions. PPI plans and methods were modified to all components being online. We describe and reflect on: initial plans and adaptation; recruitment; training and preparation; implementation, contextualisation and interpretation. Through first-hand accounts we show how the PPI processes were developed, experienced and viewed by different partners in the process. DISCUSSION AND CONCLUSIONS: While concerns have been expressed about the possible limiting effects of forgoing face-to-face contact with PPI partners, we found important benefits from the altered dynamic of the online PPI environment. There were increased opportunities for participation which might encourage the involvement of a broader demographic, and unexpected benefits in that the online platform seemed to have a 'democratising' effect on the meetings, to the benefit of the PPI processes and outcomes. Other studies may however find that their particular research context raises particular challenges for the use of online methods, especially in relation to representation and inclusion, as new barriers to participation may be raised. It is important that methodological challenges are addressed, and researchers provide detailed examples of novel methods for discussion and empirical study. PATIENT AND PUBLIC CONTRIBUTION: We report a process which involved people with lived experience of emergency conditions and members of the public. A patient member was involved in the design and implementation, and two patients with lived experience contributed to the manuscript.

2020 Annual Report of the Kansas Poison Control Center at The University of Kansas Health System.

Introduction: This is the 2020 Annual Report of the Kansas Poison Control Center (KSPCC) at The University of Kansas Health System. The KSPCC receives calls from the public, law enforcement, healthcare professionals, and public health agencies. Methods: Encounters reported to the KSPCC from January 1, 2020 through December 31, 2020 were analyzed for caller location, demographics, exposure substance, nature of exposure, route of exposure, interventions, medical outcome, and location of care. Encounters were classified as human or animal exposure, confirmed non-exposure, or information call (no exposure). Results: There were 19,780 total encounters, including 18,492 human exposure cases. These cases were primarily female (53.6%, n = 9,911) and pediatric (19 years of age or less; 59.5%, n = 10,995). Acute cases (82.7%, n = 15,294), unintentional exposures (73.8%, n = 13,643), and ingestions (85.9%, n = 15,901) were most common. The most common reported substance was household cleaning products (n = 937) in pediatric (children ≤ 5) and analgesics (n = 1,335) in adults. An increase in exposures to disinfectants and household cleaning products was seen. Moderate (n = 1,812) or major (n = 482) clinical outcomes were seen in 12.4% of cases. There were 18 deaths in 2020 reported to the KSPCC. Conclusions: Over 18,400 exposures were managed by the KSPCC in 2020. Pediatric exposures remained the most common encounter. An increase in exposures to disinfectants and other household cleaning products was seen. This report supported the continued value of the KSPCC to both public and acute healthcare in the state of Kansas.

Elevated methemoglobin levels in patients treated with hydroxocobalamin: a case series and in-vitro analysis.

BACKGROUND: Historically, the first step in treating cyanide (CN-) toxicity utilized antidotes to induce methemoglobinemia. This is concerning in patients who are already hypoxemic or have elevated carboxyhemoglobin. Hydroxocobalamin (OHCbl) is now the first-line antidote for CN- toxicity and is not known to induce methemoglobinemia. We observed elevated methemoglobin (MetHb) levels in several patients treated with OHCbl and sought to investigate the incidence of MetHb formation following administration of OHCbl. METHODS: Chart review: A single-center, retrospective case series of patients who received 5 or 10 g of hydroxocobalamin from 01/01/2011 through 04/30/2019. Data was analyzed using descriptive statistics. In-vitro study: Discarded blood was separated into whole blood and plasma samples. OHCbl and normal saline was added to reach 0×, 1×, 2×, and 4× peak therapeutic concentrations and analyzed at times 0, 2, and 4 h after administration. RESULTS: Chart review: Twenty-seven cases of OHCbl administration were identified. The median age was 53 years (IQR 38 - 64) and 20 (74.1%) were male. Exposure to a house fire or smoke inhalation was the reason for OHCbl administration in 21 (77.8%) patients. Five (18.5%) patients received 10 g of OHCbl while the rest received 5 g. Six (22.2%) patients developed methemoglobinemia, all after 5 g OHCbl administration; four had been exposed to fire and smoke, two received the medication for severe acidosis of unknown etiology not related to fire or smoke. The median peak level was 7.1% (IQR 2.2 - 16.4%) at a median time of 11.4 h post-administration. Two patients received methylene blue (MB), neither responded. Death occurred in 17 (63%) cases. In-vitro study: We observed a dose dependent elevation in total hemoglobin but did not detect any increase in MetHb. CONCLUSION: We observed a noteworthy temporal association between the formation of methemoglobinemia and the administration of hydroxocobalamin. This does not appear to be an artifact of the CO-oximeters. This could have profound implications for patients who are already hypoxemic or have impaired oxygen carrying capacity from carboxyhemoglobin.

Outcomes of acute pulmonary embolism in hospitalized patients with cancer.

BACKGROUND: Cancer-associated pulmonary embolism (PE) places a significant burden on patients and health care systems. METHODS: A retrospective cross-sectional analysis of the National Inpatient Sample (NIS) database was performed in patients with acute PE from 2002 to 2014. Among patients hospitalized with PE, we investigated the differences in clinical outcomes and healthcare utilization in patients with and without cancer. A multivariate logistic regression model was applied to calculate adjusted odds ratios (OR) to estimate the impact of cancer on clinical outcomes. Wilcoxon rank sum tests were used to determine the differences in healthcare utilization between the two cohorts. RESULTS: Among 3,313,044 patients who were discharged with a diagnosis of acute PE, 84.2% did not have cancer, while 15.8% had cancer as a comorbidity (56% metastatic cancer, 35% solid tumor without metastasis, and 9% lymphoma). Patients with cancer had a higher mean age but lower rates of common comorbidities except for coagulation deficiency than patients without a cancer diagnosis. In patients with cancer, the rate of IVC filter placement was higher (21.7% vs. 13.11%, OR 1.76 (95% CI 1.73-1.79); p < 0.0001) and thrombolytic use lower (1.34% vs. 2.15%, OR 0.68 (95% CI 0.64-0.72); p < 0.0001). Patients with cancer hospitalized for PE had a higher all-cause in-hospital mortality (11.8% vs. 6.6%, OR 1.79 (95% CI 1.75-1.83); p < 0.0001), longer length of stay (6 vs. 5 days; p < 0.0001), higher total charge per hospitalization ($30,885 vs. $27,273; p < 0.0001), and higher rates of home health services upon discharge (35.8% vs. 23.2%; p < 0.0001) compared with those without cancer. CONCLUSION: Concurrent cancer diagnosis in patients hospitalized for acute PE was associated with a 90% increase in all-cause mortality, longer length of stay, higher total charge per hospitalization, and higher rates of home health services upon discharge. The majority (56%) of patients with cancer had metastatic disease. Furthermore, there were identifiable differences in the intervention for acute PE between the two groups.

2019 Annual Report of the Kansas Poison Control Center at The University of Kansas Health System.

INTRODUCTION: This is the 2019 Annual Report of the Kansas Poison Control Center (KSPCC) at The University of Kansas Health System. The KSPCC is one of 55 certified poison control centers in the United States and serves the state of Kansas 24-hours a day, 365 days a year with certified specialists in poison information and clinical and medical toxicologists. The KSPCC receives calls from the public, law enforcement, health care professionals, and public health agencies. All calls to the KSPCC are recorded electronically in the Toxicall® data management system and uploaded in near real-time to the National Poison Data System (NPDS) which is the data repository for all poison control centers in the United States. METHODS: All encounters reported to the KSPCC from January 1, 2019 through December 31, 2019 were analyzed. Data recorded for each exposure includes caller location, age, weight, gender, exposure substance, nature of exposure, route of exposure, interventions, medical outcome, disposition, and location of care. Encounters were classified as human exposure, animal exposure, confirmed non-exposure, or information call (no exposure reported). RESULTS: The KSPCC logged 20,589 total encounters in 2019, including 19,406 human exposure cases. The KSPCC received calls from every county in Kansas. A slim majority of human exposure cases (50.5%, n = 9,790) were female. Approximately 61% (n = 11,876) of human exposures involved a child (defined as 19 years of age or less). Most encounters occurred at a residence (91.6%, n = 17,780) and most cases (64.9%, n = 12,599) originated from a residence. The majority of human exposures (85.5%, n = 16,589) were acute cases (exposures occurring over 8 hours or less). Ingestion was the most common route of exposure documented (85.3%, n = 16,548). The most commonly reported substance in pediatric (children ≤ 5) encounters was cosmetics/personal care products (n = 959) followed closely by household cleaning products (n = 943). For adult encounters, analgesics (n = 1,296) and sedative/hypnotics/antipsychotics (n = 1,084) were the most frequently involved substances. Unintentional exposures were the most common reason for exposures (75.4%, n = 14,634). Most encounters (65.9%, n = 12,780) were managed in a non-healthcare facility (i.e., a residence). Among human exposures, 14,591 involved exposures to pharmaceutical agents while 9,439 involved exposure to non-pharmaceuticals. Medical outcomes were 26.4% (n = 5,116) no effect, 18.8% (n = 3,652) minor effect, 9.3% (n = 1,813) moderate effect, and 3.1% (n = 603) major effects. There were 14 deaths in 2019 reported to the KSPCC. Cases from healthcare facilities and cases with moderate or major medical outcomes increased in 2019 compared to 2018. The number of deaths reported to the KSPCC increased in 2019 to 14 from 7 in 2018. CONCLUSIONS: The results of the 2019 Kansas Poison Control Center's annual report demonstrated that cases were received from the entire state of Kansas totaling over 19,400 human exposures per year. While pediatric exposures remained the most common encounter, there continued a trend of increasing number of cases from healthcare facilities and for cases with serious outcomes. The experience of the KSPCC is comparable to national data. This report supported the continued value of the KSPCC to both public and acute health care in the state of Kansas.

Utilization of a poison control center by critical access hospitals-one state's experience.

PURPOSE: Critical access hospitals (CAH) are an important source of exposures for poison control centers (PCC), yet there is a paucity of literature on how these calls differ from larger, more urban hospitals (UH). This study aimed to compare call characteristics from CAH and UH received by a regional PCC. METHODS: This retrospective chart review used the Toxicall® database of the Kansas PCC. All cases involving calls from a health care facility from 1 January 2012 to 31 December 2017 were identified. Cases were then identified as either from a CAH or from one of the four largest UH in Kansas for comparison. CAH were defined using the criteria from the Centers for Medicare & Medicaid Services. All information was de-identified in REDCAPS and analyzed in Microsoft Excel. FINDINGS: A total of 12,469 unique patient cases were identified, including 6392 CAH and 6077 UH cases. Patients at UH were more likely to have longer duration of medical effects and experience more severe medical effects. Significant differences in the unique exposures, rates of intentional overdose, routes, and number of exposures were found. Patients at CAH were more likely to receive activated charcoal, flumazenil, and be transferred to another healthcare facility, but were less likely to have been referred by the PCC, need an ICU admission, or require consultation with a toxicologist. CONCLUSIONS: PCC calls from CAHs were common and resulted in significant differences when compared to UH calls. Future research is warranted to optimize PCC services for rural hospitals.

2018 Annual Report of the University of Kansas Health System Poison Control Center.

INTRODUCTION: This is the 2018 Annual Report of the Kansas Poison Control Center at The University of Kansas Health System (KSPCC). The KSPCC serves the state of Kansas 24-hours per day, 365 days a year with certified specialists in poison information and clinical and medical toxicologists. METHODS: All encounters reported to the KSPCC from January 1, 2018 through December 31, 2018 were analyzed. Data recorded for each exposure included caller location, age, weight, gender, exposure substance, nature of exposure, route of exposure, interventions, medical outcome, disposition, and location of care. RESULTS: There were 21,072 total encounters, including 20,031 human exposure cases. Calls were received from every county and hospital in Kansas. Most of the exposures involved females (51.5%, n = 10,320) and a child less than 19 year of age (64%, n = 12,865). Medical outcomes were 24.5% (n = 4,912) no effect, 17.7% (n = 3,542) minor effect, 9.1% (n = 1,830) moderate effect, and 2.4% (n = 476) major effect. Seven deaths were reported in 2018. The number of exposure calls from healthcare facilities and severity of medical outcomes increased in 2018 compared to 2017. CONCLUSION: The 2018 KSPCC annual report demonstrated that the center receives calls from the entire state of Kansas totaling over 20,000 human exposures. While pediatric exposures remain the most common encounter, a trend continued of an increasing number of calls from healthcare facilities and for cases with serious outcomes. This report supported the continued value of the KSPCC to both public and acute health care in the state of Kansas.

Extended Continuous Infusion of Methylene Blue for Refractory Septic Shock.

OBJECTIVE: Highlight the utility of methylene blue (MTB) infusion for extended period for refractory vasoplegia. INTRODUCTION: Hypotension refractory to vasopressor therapy in sepsis is associated with high mortality and limited therapeutic options. Dysregulated nitric oxide (NO) pathway seems to be a major driver, and, therefore, MTB, which inhibits inducible NO synthase activity and decreases cyclic guanosine monophosphate (GMP) accumulation by directly competing with NO by binding to soluble guanylyl cyclase, has been explored. CASE DESCRIPTION: We describe a successful reversal of refractory septic shock with prolonged MTB infusion in a patient supported on multiple vasopressors at the highest clinical doses as well as venovenous extracorporeal membrane oxygenation (VV-ECMO). CONCLUSION AND CLINICAL SIGNIFICANCE: Current report suggests a potential role of MTB infusion in refractory vasoplegia even in advanced vasoplegic shock. HOW TO CITE THIS ARTICLE: Jaiswal A, Kumar M, Silver E. Extended Continuous Infusion of Methylene Blue for Refractory Septic Shock. Indian J Crit Care Med 2020;24(3):206-207.

All that glitters is not gold: Mercury poisoning in a family mimicking an infectious illness.

Three siblings with inhalational elemental mercury toxicity presented with fever, rash, and upper respiratory tract symptoms. The patients were heavily exposed to elemental mercury that was spilled in their home and then vacuumed. Initial whole blood mercury levels were elevated at >200 µg/L, 153 µg/L and 130 µg/L (Mayo Clinic Laboratories lab reference range <9 µg/L) for Cases 1, 2, and 3, respectively. All three required chelation with succimer. Clinically significant elemental mercury toxicity can resemble an infectious illness. Severe morbidity and mortality can be prevented if heavy metal poisoning is considered early, through a detailed history including an environmental exposure history. For elemental mercury spills in the home, safe and effective clean-up steps are needed. Improved public health education is needed to prevent similar household exposures.

Successful Treatment of Antihypertensive Overdose Using Intravenous Angiotensin II.

BACKGROUND: Despite multiple treatment options, antihypertensive overdose remains a cause of significant morbidity and mortality. Intravenous angiotensin II (AG II) is approved for use in vasodilatory shock. We describe 2 cases of refractory shock from antihypertensive overdose that were successfully treated using AG II. CASE REPORTS: A 24-year-old female presented after an overdose of multiple antihypertensive medications, including an angiotensin converting enzyme inhibitor (ACEI). She developed hypotension that was refractory to norepinephrine, epinephrine, and vasopressin, with a mean arterial pressure (MAP) of 57 mm Hg 9 h after emergency department arrival. Fifteen minutes after starting AG II at 10 ng/kg/min, her heart rate and MAP rose by 7 beats/min and 12 mm Hg, respectively. Her hemodynamic parameters continued to improve thereafter. She developed acute kidney injury, which resolved prior to discharge. The second patient, a 65-year-old male, presented after an overdose of multiple antihypertensive medications, including an ACEI. Despite norepinephrine, epinephrine, and hyperinsulinemia-euglycemia, he remained bradycardic and hypotensive, with a heart rate of 47 beats/min and MAP of 59 mm Hg. Thirty minutes after starting AG II at 10 ng/kg/min, his heart rate was 61 beats/min and MAP was 66 mm Hg. He recovered without apparent sequelae. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Antihypertensive overdose can lead to shock refractory to catecholamine and vasopressin therapy. Our experience suggests that AG II is efficacious in antihypertensive overdose and may be particularly efficacious in instances of ACEI overdose. However, further study is required to confirm the appropriate indication(s).

Better Together: Reliable Application of the Post-9/11 and Post-Iraq US Intelligence Tradecraft Standards Requires Collective Analysis.

Background: The events of 9/11 and the October 2002 National Intelligence Estimate on Iraq's Continuing Programs for Weapons of Mass Destruction precipitated fundamental changes within the United States Intelligence Community. As part of the reform, analytic tradecraft standards were revised and codified into a policy document - Intelligence Community Directive (ICD) 203 - and an analytic ombudsman was appointed in the newly created Office for the Director of National Intelligence to ensure compliance across the intelligence community. In this paper we investigate the untested assumption that the ICD203 criteria can facilitate reliable evaluations of analytic products. Methods: Fifteen independent raters used a rubric based on the ICD203 criteria to assess the quality of reasoning of 64 analytical reports generated in response to hypothetical intelligence problems. We calculated the intra-class correlation coefficients for single and group-aggregated assessments. Results: Despite general training and rater calibration, the reliability of individual assessments was poor. However, aggregate ratings showed good to excellent reliability. Conclusion: Given that real problems will be more difficult and complex than our hypothetical case studies, we advise that groups of at least three raters are required to obtain reliable quality control procedures for intelligence products. Our study sets limits on assessment reliability and provides a basis for further evaluation of the predictive validity of intelligence reports generated in compliance with the tradecraft standards.

XRCC1 stimulates polynucleotide kinase by enhancing its damage discrimination and displacement from DNA repair intermediates.

Human polynucleotide kinase (hPNK) is required for processing and rejoining DNA strand break termini. The 5'-DNA kinase and 3'-phosphatase activities of hPNK can be stimulated by the "scaffold" protein XRCC1, but the mechanism remains to be fully elucidated. Using a variety of fluorescence techniques, we examined the interaction of hPNK with XRCC1 and substrates that model DNA single-strand breaks. hPNK binding to substrates with 5'-OH termini was only approximately 5-fold tighter than that to identical DNA molecules with 5'-phosphate termini, suggesting that hPNK remains bound to the product of its enzymatic activity. The presence of XRCC1 did not influence the binding of hPNK to substrates with 5'-OH termini, but sharply reduced the interaction of hPNK with DNA bearing a 5'-phosphate terminus. These data, together with kinetic data obtained at limiting enzyme concentration, indicate a dual function for the interaction of XRCC1 with hPNK. First, XRCC1 enhances the capacity of hPNK to discriminate between strand breaks with 5'-OH termini and those with 5'-phosphate termini; and second, XRCC1 stimulates hPNK activity by displacing hPNK from the phosphorylated DNA product.

Epitope mapping of Ly-49G and G-like receptors: CK-1 antibody defines a polymorphic site of functional interaction with class I ligand.

Ly-49 receptors regulate mouse natural killer cell functions. Members of the polymorphic Ly-49 multigene family recognize specific alleles of major histocompatibility complex class I (MHC I) or MHC I-like proteins. Previous studies have provided insight into the nature of Ly-49A and -C interaction with their high-affinity MHC I ligands, H-2Dd and Kb, respectively. Unlike Ly-49C, recognition of MHC I by Ly-49A is regulated in part by residues within the beta4-beta5 loop of its ectodomain. Ly-49A and -G are within the same Ly-49 subfamily, and both receptors recognize Dd. However, there have been no studies that define specific sites on Ly-49G that mediate class I MHC recognition. The Ly-49G receptors of different inbred mouse strains can differ as a result of amino acid polymorphisms within their ectodomains. In this report, we have generated a novel antibody, CK-1, which recognizes Ly-49G(B6) and a Ly-49G(B6)-like receptor, Ly-49M(nonobese diabetic), but not Ly-49G(BALB/c). By exploiting the differences within ectodomains of C57BL/6 and BALB/c Ly-49G allele products, we identified epitopes recognized by the Ly-49G-specific antibodies CK-1 and Cwy-3, whose epitopes mapped within the beta4-beta5 loop and the beta1 strand, respectively, and were nonoverlapping. Although both antibodies specifically recognized the Ly-49G(B6) ectodomain, Cwy-3 was unable to block its interaction with MHC I, and CK-1 significantly inhibited it. The importance of residues within the beta4-beta5 loop in Ly-49G recognition demonstrates that its interaction with MHC I is similar to that of Ly-49A but not Ly-49C.

The rat RT1-A1c MHC molecule is a xenogeneic ligand recognized by the mouse activating Ly-49W and inhibitory Ly-49G receptors.

Mouse Ly-49 receptors are known to recognize xenogeneic ligands from hamster and rat. However, until now, there has been no description of a specific rat xenogeneic ligand for any mouse Ly-49 receptor. In this report, we identify RT1-A1c, a rat classical class I MHC molecule, as a ligand for the Ly-49G(BALB/c) inhibitory receptor and the closely related activating receptor, Ly-49W. Xenogeneic class I recognition of targets from PVG but not DA strain rats was mapped to the classical region of the RT1c haplotype by using Con A blasts from RT1c/RT1av1 intra-MHC recombinant rats as targets for RNK-16 cells expressing either Ly-49W or Ly-49G(BALB/c) receptors. Individual expression of class I molecules from PVG and DA rat strains in YB2/0 target cells demonstrate the xenogeneic recognition to be allele specific, because other class I molecules of the RT1c haplotype, RT1-A2c and RT1-U2c, and a classical class I molecule encoded by the RT1av1 haplotype, RT1-Aa, are not recognized by Ly-49W and -G(BALB/c). Furthermore, specificity for RT1-Ac can be transferred from Ly-49W to Ly-49P, which is normally unable to recognize RT1-Ac, by substitution of three residues shared by Ly-49W and -G(BALB/c) but not Ly-49P. These residues are located in the Ly-49 beta4-beta5 loop, which can determine class I allele specificity in mouse Ly-49 receptor interactions with mouse class I ligands, suggesting that mouse Ly-49 recognition of rat class I molecules follows similar principles of interaction. These findings have implications for xenotransplantation studies and for discerning Ly-49 recognition motifs present in MHC molecules.

Reciprocal transfer of class I MHC allele specificity between activating Ly-49P and Ly-49W receptors by exchange of beta 4-beta 5 loop residues.

Receptors of the Ly-49 multigene family regulate rodent NK cell functions. Ly-49Rs are highly polymorphic and exist in either activating or inhibitory forms. Examples of both Ly-49 receptor types have been shown to recognize class I MHC ligands. Ly-49Rs can distinguish between class I alleles, but the molecular basis of this discrimination is unknown. Two activating receptors, Ly-49P and Ly-49W, differ in class I recognition, recognizing H-2D(d), or H-2D(d) and D(k), respectively. In this report, we demonstrate that specificity for H-2D(k) can be transferred from Ly-49W to Ly-49P by substituting 3 aa predicted to reside in the beta4-beta5 loop of Ly-49W into Ly-49P. Replacement of these same residues of Ly-49W with corresponding residues in Ly-49P eliminates H-2D(k) recognition while still preserving H-2D(d) recognition. Further mutagenesis indicates that all 3 aa facilitate optimal class I specificity exchange. These results provide the first evidence for a specific site on Ly-49Rs, the beta4-beta5 loop, in determining class I MHC allele specificity.

Allelic variation in the ectodomain of the inhibitory Ly-49G2 receptor alters its specificity for allogeneic and xenogeneic ligands.

The Ly-49 multigene receptor family regulates mouse NK cell functions. A number of Ly-49 genes exhibit allelic variation, but the functional significance of allelic differences in extracellular domains of Ly-49 receptors regarding ligand specificity is largely unknown. Amino acid differences exist in the extracellular domains of the B6 and BALB/c allele products of the inhibitory Ly-49G receptor. We constructed chimeric Ly-49 receptors consisting of common cytoplasmic and transmembrane regions of the activating Ly-49W receptor fused with the ectodomains of the B6 and BALB/c alleles of Ly-49G. Expression of these chimeras in the RNK-16 rat NK cell line allowed us to study the specificity of inhibitory receptor ectodomains as they stimulated NK lytic activity. We found that the ectodomain of the BALB/c allele of Ly-49G recognizes both H-2D(d) and D(k) class I MHC alleles, whereas the ectodomain of the B6 allele of Ly-49G recognizes D(d), and not D(k). The specificity for D(k) as well as D(d) of the wild-type Ly-49G(BALB/c) allele product was confirmed with RNK-16 transfectants of this inhibitory receptor. Furthermore, the ectodomain of the Ly-49G(BALB/c) allele recognizes a distinct repertoire of xenogeneic ligands that only partially overlaps with that recognized by Ly-49G(B6). Our results indicate that allelic variation in Ly-49 extracellular domains can have functional significance by altering Ly-49 receptor specificity for mouse class I MHC and xenogeneic ligands.