Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy... more Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution.
To determine the importance of obtaining precontrast T1-weighted magnetization transfer (MT) MR i... more To determine the importance of obtaining precontrast T1-weighted magnetization transfer (MT) MR images for better interpretation contrast-enhanced T1-weighted MT images. One hundred fifty-five patients referred for MR imaging of the brain were examined prospectively with noncontrast T1-weighted imaging, noncontrast T1-weighted imaging with MT, contrast-enhanced T1-weighted imaging, and contrast-enhanced T1-weighted imaging with MT. In the patients who had abnormally increased signal intensity on postcontrast images (with or without MT), the four imaging sequences were evaluated with regard to number of lesions and lesional signal intensity. For each of the sequences, two experienced neuroradiologists subjectively graded the lesions on a scale of 1 to 4 (4 being the most conspicuous) with regard to abnormally increased signal intensity. Twenty-two of the 155 patients had increased signal intensity on one or more of the postcontrast sequences. Eight of these 22 patients had increased ...
Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vacci... more Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.
Proceedings of the National Academy of Sciences, 2010
Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence ... more Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/ 68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.
Journal of Geriatric Psychiatry and Neurology, 2007
In this review, we begin with a historical accounting of the evolution of the concept of mild cog... more In this review, we begin with a historical accounting of the evolution of the concept of mild cognitive dysfunction including nomenclature and criteria from Kral to Petersen. A critical analysis of the main elements relating to assessment and diagnosis of mild cognitive dysfunction are described. Methodological limitations in design, measurement, and characterization, especially as they relate to older African Americans, are identified. Data from a 15-year longitudinal study of communitydwelling, African Americans in Indianapolis indicate 23% prevalence of all-cause mild cognitive dysfunction with approximately 25% progressing to dementia in 2 years and another 25% reverting to normal in the same interval. Factors contributing to this longitudinal variability in outcome are reviewed including the role of medical health factors. We close with suggestions for next steps in the epidemiological research of mild cognitive impairment.
Children with high-risk neuroblastoma have a poor prognosis with chemotherapy alone, and hematopo... more Children with high-risk neuroblastoma have a poor prognosis with chemotherapy alone, and hematopoietic stem cell transplantation offers improved survival. As a dose-escalation strategy, tandem transplants have been used, but are associated with persistent immunocompromise. This study evaluated the provision of an autologous costimulated, activated T-cell product to support immunologic function. Nineteen subjects with high-risk neuroblastoma were enrolled in a pilot phase and 23 subjects were entered in to the randomized study. Immunologic reconstitution was defined by flow cytometric and functional assays. Next-generation sequencing was conducted to identify changes to the T-cell repertoire. Twenty-two patients were vaccinated to define effects on antibody responses. Subjects who received their autologous costimulated T-cell product on day 2 had significantly superior T-cell counts and T-cell proliferation compared with those who received T cells on day 90. Early administration of autologous T cells suppressed oligoclonality and enhanced repertoire diversity. The subjects who received the day 2 T-cell product also had better responses to the pneumococcal vaccine. The infusion of activated T cells can improve immunologic function especially when given early after transplant. This study showed the benefit of providing cell therapies during periods of maximum lymphopenia.
Background-The utility of contrast MRI for assessing myocardial viability in stable coronary arte... more Background-The utility of contrast MRI for assessing myocardial viability in stable coronary artery disease (CAD) with left ventricular dysfunction is uncertain. We therefore performed cine and contrast MRI in 24 stable patients with CAD and regional contractile abnormalities and compared MRI findings with rest-redistribution 201 Tl imaging and dobutamine echocardiography. Methods and Results-Delayed MRI contrast enhancement patterns were examined from 3 to 15 minutes after injection of 0.1 mmol/kg IV gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). Comparable MRI and 201 Tl basal and midventricular short-axis images were subdivided into 6 segments. Segments judged nonviable by quantitative and qualitative assessment of 201 Tl scans showed persistent, systematically greater MRI contrast signal intensity than segments judged viable (PՅ0.002). Delayed contrast hyperenhancement also occurred in segments judged nonviable by dobutamine echocardiography (PՅ0.03). The presence or absence of hyperenhancement correlated most closely with nonviability and viability, respectively, in segments that were akinetic or dyskinetic under resting conditions (83% concordance with 201 Tl in both cases). In segments with resting hypokinesis, 58% of segments showing hyperenhancement were judged viable by 201 Tl and may have represented an admixture of scar tissue and viable myocardium. Conclusions-Delayed (by 3 to 15 minutes) hyperenhancement of Gd-DTPA contrast-enhanced MRI images occurs frequently in dysfunctional areas of the left ventricle in patients with stable CAD. Hyperenhancement is associated with nonviability by rest-redistribution 201 Tl scintigraphy and dobutamine echocardiography, particularly in regions exhibiting resting akinesis/dyskinesis. The absence of hyperenhancement correlates with radionuclide and echocardiographic determinations of viability, regardless of resting contractile function. (Circulation. 1998;98:2687-2694.)
The risk of Epstein-Barr virus lymphoproliferative disease (EBV-LPD) increases with the use of hi... more The risk of Epstein-Barr virus lymphoproliferative disease (EBV-LPD) increases with the use of highly immunosuppressive therapies. Allogeneic BMT, especially supported by T-cell-depleted stem cell products, is a risk factor for EBV-LPD. Although the risk of EBV-LPD after autologous transplantation is low, case reports of this complication in the autologous setting exist. We report a higher incidence than previously described of EBV-LPD in children undergoing sequential high-dose chemotherapy supported with CD34 selected peripheral blood stem cells (CD34 þ PBSC). The median time to LPD after tandem transplant was 3 months (range 1-5 months). Five patients out of 156 (3.5%) developed EBV-LPD while enrolled on two trials of tandem autologous SCT in high-risk pediatric malignancies. Both studies employed five cycles of induction therapy, followed by tandem autologous PBSC transplants. In all, 108 out of 156 patients received CD34 þ PBSC; 48 received unselected PBSC. All patients contracting LPD were from the CD34 selected group. Treatment of EBV-LPD included rituximab in four out of five patients, IVIg in two out of five patients, and gancyclovir in two out of five patients. EBV-LPD resolved in four out of five patients. We conclude that the combination of tandem SCT and CD34 selection may have increased immunosuppression in these patients to a point where there is an elevated risk of EBV-LPD.
Abstract This 1978???1982 update of an earlier study examined the 1946???1982 mortality experienc... more Abstract This 1978???1982 update of an earlier study examined the 1946???1982 mortality experience of 16 661 man-made mineral fibre workers employed 1 yr or more (6 months for two plants) during 1945???1963 (1940???1963 for one plant) at one or more of 17 US ...
A medical surveillance program and epidemiologic study of 408 former workers of the Drake Chemica... more A medical surveillance program and epidemiologic study of 408 former workers of the Drake Chemical Company (now a Superfund waste site) was established in 1986. The Drake Health Registry Study was initiated because these workers had probable past exposures to beta-naphthylamine (BNA), a potent bladder carcinogen. The registry is widely viewed as a model for notification of workers at high risk of disease due to past occupational exposures. By the 40th month, 90% of the 366 living workers had been notified of the existence of the registry; 262 had been enrolled in the annual or semi-annual screening for bladder cancer. Among these, 27 persons have had abnormal screening results indicating moderate to high risk of bladder cancer and have been made eligible for further diagnostic tests. While no invasive bladder tumors were found among 18 persons completing the extended diagnostic evaluation, two diagnoses of moderate to severe dysplasia have been made. The registry has also identified three living and three deceased cases of bladder cancer in the cohort; a mortality analysis showed a 20- to 30-fold excess of bladder cancer. An incidence projection, based on the six identified cases, reveals that between six and ten new bladder cancer cases are likely to occur among the Drake cohort over the next 20 year period.
Double-stranded DNA cleavage of light-activated lysine conjugates is strongly enhanced at the sli... more Double-stranded DNA cleavage of light-activated lysine conjugates is strongly enhanced at the slightly acidic pH (<7) suitable for selective targeting of cancer cells. This enhancement stems from the presence of two amino groups of different basicities. The first amino group plays an auxiliary role by enhancing solubility and affinity to DNA whereas the second amino group which is positioned next to the light-activated DNA-cleaver undergoes protonation at the desired pH threshold.
Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy... more Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution.
To determine the importance of obtaining precontrast T1-weighted magnetization transfer (MT) MR i... more To determine the importance of obtaining precontrast T1-weighted magnetization transfer (MT) MR images for better interpretation contrast-enhanced T1-weighted MT images. One hundred fifty-five patients referred for MR imaging of the brain were examined prospectively with noncontrast T1-weighted imaging, noncontrast T1-weighted imaging with MT, contrast-enhanced T1-weighted imaging, and contrast-enhanced T1-weighted imaging with MT. In the patients who had abnormally increased signal intensity on postcontrast images (with or without MT), the four imaging sequences were evaluated with regard to number of lesions and lesional signal intensity. For each of the sequences, two experienced neuroradiologists subjectively graded the lesions on a scale of 1 to 4 (4 being the most conspicuous) with regard to abnormally increased signal intensity. Twenty-two of the 155 patients had increased signal intensity on one or more of the postcontrast sequences. Eight of these 22 patients had increased ...
Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vacci... more Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.
Proceedings of the National Academy of Sciences, 2010
Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence ... more Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/ 68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.
Journal of Geriatric Psychiatry and Neurology, 2007
In this review, we begin with a historical accounting of the evolution of the concept of mild cog... more In this review, we begin with a historical accounting of the evolution of the concept of mild cognitive dysfunction including nomenclature and criteria from Kral to Petersen. A critical analysis of the main elements relating to assessment and diagnosis of mild cognitive dysfunction are described. Methodological limitations in design, measurement, and characterization, especially as they relate to older African Americans, are identified. Data from a 15-year longitudinal study of communitydwelling, African Americans in Indianapolis indicate 23% prevalence of all-cause mild cognitive dysfunction with approximately 25% progressing to dementia in 2 years and another 25% reverting to normal in the same interval. Factors contributing to this longitudinal variability in outcome are reviewed including the role of medical health factors. We close with suggestions for next steps in the epidemiological research of mild cognitive impairment.
Children with high-risk neuroblastoma have a poor prognosis with chemotherapy alone, and hematopo... more Children with high-risk neuroblastoma have a poor prognosis with chemotherapy alone, and hematopoietic stem cell transplantation offers improved survival. As a dose-escalation strategy, tandem transplants have been used, but are associated with persistent immunocompromise. This study evaluated the provision of an autologous costimulated, activated T-cell product to support immunologic function. Nineteen subjects with high-risk neuroblastoma were enrolled in a pilot phase and 23 subjects were entered in to the randomized study. Immunologic reconstitution was defined by flow cytometric and functional assays. Next-generation sequencing was conducted to identify changes to the T-cell repertoire. Twenty-two patients were vaccinated to define effects on antibody responses. Subjects who received their autologous costimulated T-cell product on day 2 had significantly superior T-cell counts and T-cell proliferation compared with those who received T cells on day 90. Early administration of autologous T cells suppressed oligoclonality and enhanced repertoire diversity. The subjects who received the day 2 T-cell product also had better responses to the pneumococcal vaccine. The infusion of activated T cells can improve immunologic function especially when given early after transplant. This study showed the benefit of providing cell therapies during periods of maximum lymphopenia.
Background-The utility of contrast MRI for assessing myocardial viability in stable coronary arte... more Background-The utility of contrast MRI for assessing myocardial viability in stable coronary artery disease (CAD) with left ventricular dysfunction is uncertain. We therefore performed cine and contrast MRI in 24 stable patients with CAD and regional contractile abnormalities and compared MRI findings with rest-redistribution 201 Tl imaging and dobutamine echocardiography. Methods and Results-Delayed MRI contrast enhancement patterns were examined from 3 to 15 minutes after injection of 0.1 mmol/kg IV gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA). Comparable MRI and 201 Tl basal and midventricular short-axis images were subdivided into 6 segments. Segments judged nonviable by quantitative and qualitative assessment of 201 Tl scans showed persistent, systematically greater MRI contrast signal intensity than segments judged viable (PՅ0.002). Delayed contrast hyperenhancement also occurred in segments judged nonviable by dobutamine echocardiography (PՅ0.03). The presence or absence of hyperenhancement correlated most closely with nonviability and viability, respectively, in segments that were akinetic or dyskinetic under resting conditions (83% concordance with 201 Tl in both cases). In segments with resting hypokinesis, 58% of segments showing hyperenhancement were judged viable by 201 Tl and may have represented an admixture of scar tissue and viable myocardium. Conclusions-Delayed (by 3 to 15 minutes) hyperenhancement of Gd-DTPA contrast-enhanced MRI images occurs frequently in dysfunctional areas of the left ventricle in patients with stable CAD. Hyperenhancement is associated with nonviability by rest-redistribution 201 Tl scintigraphy and dobutamine echocardiography, particularly in regions exhibiting resting akinesis/dyskinesis. The absence of hyperenhancement correlates with radionuclide and echocardiographic determinations of viability, regardless of resting contractile function. (Circulation. 1998;98:2687-2694.)
The risk of Epstein-Barr virus lymphoproliferative disease (EBV-LPD) increases with the use of hi... more The risk of Epstein-Barr virus lymphoproliferative disease (EBV-LPD) increases with the use of highly immunosuppressive therapies. Allogeneic BMT, especially supported by T-cell-depleted stem cell products, is a risk factor for EBV-LPD. Although the risk of EBV-LPD after autologous transplantation is low, case reports of this complication in the autologous setting exist. We report a higher incidence than previously described of EBV-LPD in children undergoing sequential high-dose chemotherapy supported with CD34 selected peripheral blood stem cells (CD34 þ PBSC). The median time to LPD after tandem transplant was 3 months (range 1-5 months). Five patients out of 156 (3.5%) developed EBV-LPD while enrolled on two trials of tandem autologous SCT in high-risk pediatric malignancies. Both studies employed five cycles of induction therapy, followed by tandem autologous PBSC transplants. In all, 108 out of 156 patients received CD34 þ PBSC; 48 received unselected PBSC. All patients contracting LPD were from the CD34 selected group. Treatment of EBV-LPD included rituximab in four out of five patients, IVIg in two out of five patients, and gancyclovir in two out of five patients. EBV-LPD resolved in four out of five patients. We conclude that the combination of tandem SCT and CD34 selection may have increased immunosuppression in these patients to a point where there is an elevated risk of EBV-LPD.
Abstract This 1978???1982 update of an earlier study examined the 1946???1982 mortality experienc... more Abstract This 1978???1982 update of an earlier study examined the 1946???1982 mortality experience of 16 661 man-made mineral fibre workers employed 1 yr or more (6 months for two plants) during 1945???1963 (1940???1963 for one plant) at one or more of 17 US ...
A medical surveillance program and epidemiologic study of 408 former workers of the Drake Chemica... more A medical surveillance program and epidemiologic study of 408 former workers of the Drake Chemical Company (now a Superfund waste site) was established in 1986. The Drake Health Registry Study was initiated because these workers had probable past exposures to beta-naphthylamine (BNA), a potent bladder carcinogen. The registry is widely viewed as a model for notification of workers at high risk of disease due to past occupational exposures. By the 40th month, 90% of the 366 living workers had been notified of the existence of the registry; 262 had been enrolled in the annual or semi-annual screening for bladder cancer. Among these, 27 persons have had abnormal screening results indicating moderate to high risk of bladder cancer and have been made eligible for further diagnostic tests. While no invasive bladder tumors were found among 18 persons completing the extended diagnostic evaluation, two diagnoses of moderate to severe dysplasia have been made. The registry has also identified three living and three deceased cases of bladder cancer in the cohort; a mortality analysis showed a 20- to 30-fold excess of bladder cancer. An incidence projection, based on the six identified cases, reveals that between six and ten new bladder cancer cases are likely to occur among the Drake cohort over the next 20 year period.
Double-stranded DNA cleavage of light-activated lysine conjugates is strongly enhanced at the sli... more Double-stranded DNA cleavage of light-activated lysine conjugates is strongly enhanced at the slightly acidic pH (<7) suitable for selective targeting of cancer cells. This enhancement stems from the presence of two amino groups of different basicities. The first amino group plays an auxiliary role by enhancing solubility and affinity to DNA whereas the second amino group which is positioned next to the light-activated DNA-cleaver undergoes protonation at the desired pH threshold.
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Papers by C. Callahan