Comparison of Toxicities among Different Bumped Kinase Inhibitor Analogs for Treatment of Cryptosporidiosis.

Recent advances on the development of bumped kinase inhibitors for treatment of cryptosporidiosis have focused on the 5-aminopyrazole-4-carboxamide scaffold, due to analogs that have less hERG inhibition, superior efficacy, and strong in vitro safety profiles. Three compounds, BKI-1770, -1841, and -1708, showed strong efficacy in C. parvum infected mice. Both BKI-1770 and BKI-1841 had efficacy in the C. parvum newborn calf model, reducing diarrhea and oocyst excretion. However, both compounds caused hyperflexion of the limbs seen as dropped pasterns. Toxicity experiments in rats and calves dosed with BKI-1770 showed enlargement of the epiphyseal growth plate at doses only slightly higher than the efficacious dose. Mice were used as a screen to check for bone toxicity, by changes to the tibia epiphyseal growth plate, or neurological causes, by use of a locomotor activity box. These results showed neurological effects from both BKI-1770 and BKI-1841 and bone toxicity in mice from BKI-1770, indicating one or both effects may be contributing to toxicity. However, BKI-1708 remains a viable treatment candidate for further evaluation as it showed no signs of bone toxicity or neurological effects in mice.

Bumped Kinase Inhibitors as therapy for apicomplexan parasitic diseases: lessons learned.

Bumped Kinase Inhibitors, targeting Calcium-dependent Protein Kinase 1 in apicomplexan parasites with a glycine gatekeeper, are promising new therapeutics for apicomplexan diseases. Here we will review advances, as well as challenges and lessons learned regarding efficacy, safety, and pharmacology that have shaped our selection of pre-clinical candidates.

Development of 5-Aminopyrazole-4-carboxamide-based Bumped-Kinase Inhibitors for Cryptosporidiosis Therapy.

Cryptosporidium is a leading cause of pediatric diarrhea worldwide. Currently, there is neither a vaccine nor a consistently effective drug available for this disease. Selective 5-aminopyrazole-4-carboxamide-based bumped-kinase inhibitors (BKIs) are effective in both in vitro and in vivo models of Cryptosporidium parvum. Potential cardiotoxicity in some BKIs led to the continued exploration of the 5-aminopyrazole-4-carboxamide scaffold to find safe and effective drug candidates for Cryptosporidium. A series of newly designed BKIs were tested for efficacy against C. parvum using in vitro and in vivo (mouse infection model) assays and safety issues. Compound 6 (BKI 1708) was found to be efficacious at 8 mg/kg dosed once daily (QD) for 5 days with no observable signs of toxicity up to 200 mg/kg dosed QD for 7 days. Compound 15 (BKI 1770) was found to be efficacious at 30 mg/kg dosed twice daily (BID) for 5 days with no observable signs of toxicity up to 300 mg/kg dosed QD for 7 days. Compounds 6 and 15 are promising preclinical leads for cryptosporidiosis therapy with acceptable safety parameters and efficacy in the mouse model of cryptosporidiosis.

Advances in bumped kinase inhibitors for human and animal therapy for cryptosporidiosis.

Improvements have been made to the safety and efficacy of bumped kinase inhibitors, and they are advancing toward human and animal use for treatment of cryptosporidiosis. As the understanding of bumped kinase inhibitor pharmacodynamics for cryptosporidiosis therapy has increased, it has become clear that better compounds for efficacy do not necessarily require substantial systemic exposure. We now have a bumped kinase inhibitor with reduced systemic exposure, acceptable safety parameters, and efficacy in both the mouse and newborn calf models of cryptosporidiosis. Potential cardiotoxicity is the limiting safety parameter to monitor for this bumped kinase inhibitor. This compound is a promising pre-clinical lead for cryptosporidiosis therapy in animals and humans.

Human rhinovirus detection in the lower respiratory tract of hematopoietic cell transplant recipients: association with mortality.

Human rhinoviruses are the most common respiratory viruses detected in patients after hematopoietic cell transplantation. Although rhinovirus appears to occasionally cause severe lower respiratory tract infection in immunocompromised patients, the clinical significance of rhinovirus detection in the lower respiratory tract remains unknown. We evaluated 697 recipients transplanted between 1993 and 2015 with rhinovirus in respiratory samples. As comparative cohorts, 273 recipients with lower respiratory tract infection caused by respiratory syncytial virus (N=117), parainfluenza virus (N=120), or influenza (N=36) were analyzed. Factors associated with mortality were analyzed using Cox proportional hazard models. Among 569 subjects with rhinovirus upper respiratory tract infection and 128 subjects with rhinovirus lower respiratory tract infection, probabilities of overall mortality at 90 days were 6% and 41%, respectively (P<0.001). The survival rate after lower respiratory tract infection was not affected by the presence of co-pathogens (55% in patients with co-pathogens, 64% in patients without, P=0.34). Low monocyte count (P=0.027), oxygen use (P=0.015), and steroid dose greater than 1 mg/kg/day (P=0.003) before diagnosis were significantly associated with mortality among patients with lower respiratory tract infection in multivariable analysis. Mortality after rhinovirus lower respiratory tract infection was similar to that after lower respiratory tract infection by respiratory syncytial virus, parainfluenza virus or influenza in an adjusted model. In summary, transplant recipients with rhinovirus detection in the lower respiratory tract had high mortality rates comparable to viral pneumonia associated with other well-established respiratory viruses. Our data suggest rhinovirus can contribute to severe pulmonary disease in immunocompromised hosts.

Idiopathic pneumonia syndrome after hematopoietic cell transplantation: evidence of occult infectious etiologies.

Newer diagnostic methods may link more idiopathic pneumonia syndrome (IPS) cases to an infectious agent. Bronchoalveolar lavage (BAL) samples from 69 hematopoietic cell transplant (HCT) recipients with IPS diagnosed between 1992 and 2006 were tested for 28 pathogens (3 bacteria and 25 viruses) by quantitative polymerase chain reaction and for Aspergillus by galactomannan assay. Research BALs from 21 asymptomatic HCT patients served as controls. Among 69 HCT patients with IPS, 39 (56.5%) had a pathogen detected. The most frequent pathogens were human herpesvirus-6 (HHV-6) (N = 20 [29%]) followed by human rhinovirus (HRV), cytomegalovirus (CMV), and Aspergillus (N = 8 [12%] in each). HHV-6 and HRV were rarely detected in controls, whereas CMV and Aspergillus were occasionally detected with low pathogen load. Patients with pathogens had worse day-100 survival than those without (hazard ratio, 1.88; P = .03). Mortality in patients with only pathogens of "uncertain" significance in lung was similar to that in patients with pathogens of "established" significance. Metagenomic next-generation sequencing did not reveal additional significant pathogens. Our study demonstrated that approximately half of patients with IPS had pathogens detected in BAL, and pathogen detection was associated with increased mortality. Thus, an expanded infection detection panel can significantly increase the diagnostic precision for idiopathic pneumonia.

Langerhans cell homeostasis and turnover after nonmyeloablative and myeloablative allogeneic hematopoietic cell transplantation.

BACKGROUND: Langerhans cells (LCs) are self-renewing epidermal myeloid cells that can migrate and mature into dendritic cells. Recipient LCs that survive cytotoxic therapy given in preparation for allogeneic hematopoietic cell transplantation may prime donor T cells to mediate cutaneous graft-versus-host disease (GVHD). This possible association, however, has not been investigated in the setting of nonmyeloablative allografting. METHODS: We prospectively studied the kinetics of LC-chimerism after sex-mismatched allogeneic hematopoietic cell transplantation with nonmyeloablative (n=23) or myeloablative (n=25) conditioning. Combined XY-FISH and Langerin-staining was used to assess donor LC-chimerism in skin biopsies obtained on days 28, 56, and 84 after transplant. The degree of donor LC-chimerism was correlated with the development of skin GVHD. RESULTS: We observed significantly delayed donor LC-engraftment after nonmyeloablative transplantation compared with other hematopoietic compartments and compared with LC-engraftment after myeloablative conditioning. In most recipients of nonmyeloablative transplants, recipient LCs proliferated in situ, recruitment of donor-LCs was delayed by two months, and full donor LC-chimerism was only reached by day 84 after transplant. Although persistence of host LCs on day-28 after transplant was not predictive for acute or chronic skin GVHD, the recruitment of donor-derived LCs was associated with nonspecific inflammatory infiltrates (P=0.009). CONCLUSIONS: These results show that LCs can self-renew locally but are replaced by circulating precursors even after minimally toxic nonmyeloablative transplant conditioning. Cutaneous inflammation accompanies donor LC-engraftment, but differences in LC conversion-kinetics do not predict clinical or histopathological GVHD.

Blood and gastric FOXP3+ T cells are not decreased in human gastric graft-versus-host disease.

Previous studies suggest regulatory T cells (Tregs) inhibit graft-versus-host disease (GVHD) in mouse and human hematopoietic cell transplant (HCT) recipients. As the gastrointestinal tract represents one of the most common and severe sites of GVHD-related tissue damage, we sought to determine whether a deficit in circulating or gastric mucosal Treg numbers correlates with the clinical onset of gastric GVHD. We used the marker FOXP3 to quantify Tregs in blood and in gastric antral biopsies in a cohort of 60 allogeneic HCT recipients undergoing endoscopy at a single center to evaluate symptoms suspicious for gastrointestinal GVHD. We show for the first time in the gastric mucosa and, contrary to existing reports, in the blood, that the percent of T cells expressing FOXP3 is at least as high in the presence as in the absence of GVHD involving the upper gut. There was no correlation of Treg frequency with the histologic or clinical severity of gastrointestinal GVHD. We conclude that Treg depletion is not a central feature in the pathogenesis of gastric GVHD in humans.

Refractory colitis following anti-CTLA4 antibody therapy: analysis of mucosal FOXP3+ T cells.

Ipilimumab is a humanized antibody to CTLA4 and is used to treat cancers refractory to conventional treatment. We treated 21 patients with refractory melanoma or prostate cancer with anti-CTLA4 antibody (ipilimumab), with subsequent development of significant colitis in nine cases. Two of these nine did not respond rapidly to high-dose (2 mg kg(-1) day(-1)) glucocorticoids or infliximab. They required additional immunosuppression, and one ultimately died of opportunistic infection, representing a more refractory course than has previously been described complicating ipilimumab therapy. Both patients had received radiation to the pelvis for prostate cancer less than 1 year prior to receiving ipilimumab. We performed immunohistochemical analysis of colon biopsies from ipilimumab recipients to determine if colitis correlates with depletion of intramucosal FOXP3(+) regulatory T cells (Tregs), which normally express CTLA4. However, we found no evidence of FOXP3(+) T cell depletion in any of the nine patients who developed colitis.

Safety and immunologic effects of IL-15 administration in nonhuman primates.

The administration of cytokines that modulate endogenous or transferred T-cell immunity could improve current approaches to clinical immunotherapy. Interleukin-2 (IL-2) is used most commonly for this purpose, but causes systemic toxicity and preferentially drives the expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cells, which can inhibit antitumor immunity. IL-15 belongs to the gamma(c) cytokine family and possesses similar properties to IL-2, including the ability to induce T-cell proliferation. Whereas IL-2 promotes apoptosis and limits the survival of CD8(+) memory T cells, IL-15 is required for the establishment and maintenance of CD8(+) T-cell memory. However, limited data are available to guide the clinical use of IL-15. Here, we demonstrate in nonhuman primates that IL-15 administration expands memory CD8(+) and CD4(+) T cells, and natural killer (NK) cells in the peripheral blood, with minimal increases in CD4(+)CD25(+)Foxp3(+) regulatory T cells. Daily administration of IL-15 resulted in persistently elevated plasma IL-15 levels and transient toxicity. Intermittent administration of IL-15 allowed clearance of IL-15 between doses and was safe for more than 3 weeks. These findings demonstrate that IL-15 has profound immunomodulatory properties distinct from those described for IL-2, and suggest that intermittent administration of IL-15 should be considered in clinical studies.

Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid.

BACKGROUND: The diagnosis of invasive pulmonary aspergillosis (IPA) remains challenging. Culture and histopathological examination of bronchoalveolar lavage (BAL) fluid are useful but have suboptimal sensitivity and in the case of culture may require several days for fungal growth to be evident. Detection of Aspergillus DNA in BAL fluid by quantitative PCR (qPCR) offers the potential for earlier diagnosis and higher sensitivity. It is important to adopt quality control measures in PCR assays to address false positives and negatives which can hinder accurate evaluation of diagnostic performance. METHODS: BAL fluid from 94 episodes of pneumonia in 81 patients was analyzed. Thirteen episodes were categorized as proven or probable IPA using Mycoses Study Group criteria. The pellet and the supernatant fractions of the BAL were separately assayed. A successful extraction was confirmed with a human 18S rRNA gene qPCR. Inhibition in each qPCR was measured using an exogenous DNA based internal amplification control (IAC). The presence of DNA from pathogens in the Aspergillus genus was detected using qPCR targeting fungal 18S rRNA gene. RESULTS: Human 18S rRNA gene qPCR confirmed successful DNA extraction of all samples. IAC detected some degree of initial inhibition in 11 samples. When culture was used to diagnose IPA, the sensitivity and specificity were 84.5% and 100% respectively. Receiver-operating characteristic analysis of qPCR showed that a cutoff of 13 fg of Aspergillus genomic DNA generated a sensitivity, specificity, positive and negative predictive value of 77%, 88%, 50%, 96% respectively. BAL pellet and supernatant analyzed together resulted in sensitivity and specificity similar to BAL pellet alone. Some patients did not meet standard criteria for IPA, but had consistently high levels of Aspergillus DNA in BAL fluid by qPCR. CONCLUSION: The Aspergillus qPCR assay detected Aspergillus DNA in 76.9% of subjects with proven or probable IPA when the concentrated BAL fluid pellet fraction was used for diagnosis. There was no benefit from analyzing the BAL supernatant fraction. Use of both extraction and amplification controls provided optimal quality control for interpreting qPCR results and therefore may increase our understanding of the true potential of qPCR for the diagnosis of IPA.

Tissue inhibitor of metalloproteinase-1 moderates airway re-epithelialization by regulating matrilysin activity.

Obliterative bronchiolitis (OB) is the histopathological finding in chronic lung allograft rejection. Mounting evidence suggests that epithelial damage drives the development of airway fibrosis in OB. Tissue inhibitor of metalloproteinase (TIMP)-1 expression increases in lung allografts and is associated with the onset of allograft rejection. Furthermore, in a mouse model of OB, airway obliteration is reduced in TIMP-1-deficient mice. Matrilysin (matrix metallproteinase-7) is essential for airway epithelial repair and is required for the re-epithelialization of airway wounds by facilitating cell migration; therefore, the goal of this study was to determine whether TIMP-1 inhibits re-epithelialization through matrilysin. We found that TIMP-1 and matrilysin co-localized in the epithelium of human lungs with OB and both co-localized and co-immunoprecipitated in wounded primary airway epithelial cultures. TIMP-1-deficient cultures migrated faster, and epithelial cells spread to a greater extent compared with wild-type cultures. TIMP-1 also inhibited matrilysin-mediated cell migration and spreading in vitro. In vivo, TIMP-1 deficiency enhanced airway re-epithelialization after naphthalene injury. Furthermore, TIMP-1 and matrilysin co-localized in airway epithelial cells adjacent to the wound edge. Our data demonstrate that TIMP-1 interacts with matrix metalloproteinases and regulates matrilysin activity during airway epithelial repair. Furthermore, we speculate that TIMP-1 overexpression restricts airway re-epithelialization by inhibiting matrilysin activity, contributing to a stereotypic injury response that promotes airway fibrosis via bronchiole airway epithelial damage and obliteration.

MyD88 signaling contributes to early pulmonary responses to Aspergillus fumigatus.

Toll-like receptors and the beta-glucan receptor, dectin-1, mediate macrophage inflammatory responses to Aspergillus fumigatus through MyD88-dependent and -independent signaling mechanisms; however, pulmonary inflammatory responses in MyD88-deficient mice challenged with A. fumigatus are poorly defined. The role of MyD88 signaling in early pulmonary inflammation and fungal clearance was evaluated in C57BL/6J wild-type (WT) and MyD88-deficient (MyD88-/-) mice. Early (<48 h) after infection, MyD88-/- mice had higher fungal burdens than those of WT mice, although fungal burdens rapidly declined (>72 h) in both. MyD88-/- mice had less consolidated inflammation, with fewer NK cells, in lung tissue early (24 h) after infection than did WT mice. At the latter time point, MyD88-/- mouse lungs were characterized by a large amount of necrotic cellular debris and fibrin, while WT lungs had organized inflammation. Although there were equivalent numbers of macrophages in WT and MyD88-/- mouse lung tissues, MyD88-/- cells demonstrated delayed uptake of green fluorescent protein-expressing A. fumigatus (GFP-Af293); histologically, MyD88-/- mouse lungs had more hyphal invasion of terminal airways and vessels, the appearance of bronchiolar epithelial cell necrosis, and necrotizing vasculitis. MyD88-/- lung homogenates contained comparatively decreased amounts of interleukin-1beta (IL-1beta), IL-6, KC, and gamma interferon and paradoxically increased amounts of tumor necrosis factor alpha and macrophage inflammatory protein 1alpha. These data indicate that the MyD88-dependent pathway mediates acute pulmonary fungal clearance, inflammation, and tissue injury very early after infection. Resolution of abnormalities within a 3-day window demonstrates the importance of redundant signaling pathways in mediating pulmonary inflammatory responses to fungi.

Quantitative real-time polymerase chain reaction for detection of adenovirus after T cell-replete hematopoietic cell transplantation: viral load as a marker for invasive disease.

BACKGROUND: The value of adenovirus plasma DNA detection as an indicator for adenovirus disease is unknown in the context of T cell-replete hematopoietic cell transplantation, of which adenovirus disease is an uncommon but serious complication. METHODS: Three groups of 62 T cell-replete hematopoietic cell transplant recipients were selected and tested for adenovirus in plasma by polymerase chain reaction. RESULTS: Adenovirus was detected in 21 (87.5%) of 24 patients with proven adenovirus disease (group 1), in 4 (21%) of 19 patients who shed adenovirus (group 2), and in 1 (10.5%) of 19 uninfected control patients. The maximum viral load was significantly higher in group 1 (median maximum viral load, 6.3x10(6) copies/mL; range, 0 to 1.0x10(9) copies/mL) than in group 2 (median maximum viral load, 0 copies/mL; range, 0 to 1.7x10(8) copies/mL; P<.001) and in group 3 (median maximum viral load, 0 copies/mL; range 0-40 copies/mL; P<.001). All patients in group 2 who developed adenoviremia had symptoms compatible with adenovirus disease (i.e., possible disease). A minimal plasma viral load of 10(3) copies/mL was detected in all patients with proven or possible disease. Adenoviremia was detectable at a median of 19.5 days (range, 8-48 days) and 24 days (range, 9-41 days) before death for patients with proven and possible adenovirus disease, respectively. CONCLUSION: Sustained or high-level adenoviremia appears to be a specific and sensitive indicator of adenovirus disease after T cell-replete hematopoietic cell transplantation. In the context of low prevalence of adenovirus disease, the use of polymerase chain reaction of plasma specimens to detect virus might be a valuable tool to identify and treat patients at risk for viral invasive disease.

Tumor paint: a chlorotoxin:Cy5.5 bioconjugate for intraoperative visualization of cancer foci.

Toward the goal of developing an optical imaging contrast agent that will enable surgeons to intraoperatively distinguish cancer foci from adjacent normal tissue, we developed a chlorotoxin:Cy5.5 (CTX:Cy5.5) bioconjugate that emits near-IR fluorescent signal. The probe delineates malignant glioma, medulloblastoma, prostate cancer, intestinal cancer, and sarcoma from adjacent non-neoplastic tissue in mouse models. Metastatic cancer foci as small as a few hundred cells were detected in lymph channels. Specific binding to cancer cells is facilitated by matrix metalloproteinase-2 (MMP-2) as evidenced by reduction of CTX:Cy5.5 binding in vitro and in vivo by a pharmacologic blocker of MMP-2 and induction of CTX:Cy5.5 binding in MCF-7 cells following transfection with a plasmid encoding MMP-2. Mouse studies revealed that CTX:Cy5.5 has favorable biodistribution and toxicity profiles. These studies show that CTX:Cy5.5 has the potential to fundamentally improve intraoperative detection and resection of malignancies.

Respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection.

The incidence of respiratory virus infection after hematopoietic cell transplantation (HCT) has probably been underestimated with conventional testing methods in symptomatic patients. This prospective study assessed viral infection episodes by testing weekly respiratory samples collected from HCT recipients, with and without symptoms reported by questionnaire, for 100 days after HCT. Samples were tested by culture and direct fluorescent antibody testing for respiratory syncytial virus (RSV), parainfluenza virus (PIV), and influenza A and B, and by quantitative reverse transcription-polymerase chain reaction for RSV, PIV, influenza A and B, and metapneumovirus (MPV). Of 122 patients, 30 (25%) had 32 infection episodes caused by RSV (5), PIV (17), MPV (6), influenza (3), RSV, or influenza (1). PIV, with a cumulative incidence estimate of 17.9%, was the only virus for which asymptomatic infection was detected. Lower virus copy number in patients with no or one symptom compared with 2 or more symptoms was found for all viruses in all patients (P < .001), with PIV infection having a similar virus-specific comparison (P = .004). Subclinical infection with PIV may help explain why infection-control programs that emphasize symptoms are effective against RSV and influenza but often not against PIV.

Marrow fibrosis as a risk factor for posttransplantation outcome in patients with advanced myelodysplastic syndrome or acute myeloid leukemia with multilineage dysplasia.

Marrow fibrosis is considered a poor prognostic factor in patients with myelodysplastic syndrome (MDS). The affect of fibrosis on outcomes after hematopoietic cell transplantation (HCT) in patients with MDS has not been examined. We performed a retrospective analysis in 471 patients with MDS or acute myeloid leukemia with multilineage dysplasia arising from MDS, 113 with and 358 without marrow fibrosis, who received myeloablative allogeneic HCT. Post-HCT follow-up was 0.3-10 years (median, 3.6 years) for patients with, and 0.6-12 years (median, 5 years) for patients without fibrosis. Engraftment was significantly delayed in patients with fibrosis (hazard ratio [HR] = 0.4; P < .001). Overall, there were no significant differences in overall survival (OS), relapse-free survival (RFS), and nonrelapse mortality (NRM) between patients with and without fibrosis. However, among patients with advanced disease (int-2 or high-risk disease by the International Prognostic Scoring System), OS (P = .03), RFS (P = .04), and NRM (P = .04) were inferior when marrow fibrosis was present. Given that marrow fibrosis is a poor prognostic factor for patients with MDS, and that it does not appear to affect outcome of transplantation in patients with earlier-stage disease but has a negative impact on outcome for patients with advanced disease, patients with earlier-stage MDS and marrow fibrosis might be considered for HCT earlier than their disease stage would normally dictate.

Reduced expression of inducible gelatinase B/matrix metalloproteinase-9 in monocytes from patients with myelodysplastic syndrome: Correlation of inducible levels with the percentage of cytogenetically marked cells and with marrow cellularity.

Regulatory molecules produced by stromal cells are often membrane bound until cleaved by matrix metalloproteinases (MMPs); cleavage can either activate or inactivate regulatory functions. We report here that marrow stromal cells induce the expression of MMP-9 in monocytes. Induction was contact independent and could be reproduced with recombinant MCP-1/CCL2, whereas IL-6, M-CSF, G-CSF, GM-CSF, IL-8/CXCL8, SDF-1/CXCL12, and MGSA/CXCL1 did not have this effect. Stroma-induced levels of MMP-9 in the monocyte population from healthy donors were relatively consistent, whereas induced levels varied significantly (P < .001) in the CD14+ population from 27 patients with myelodysplastic syndrome (MDS). In patients with a clonal chromosomal marker, the level of inducible MMP-9 expression in the monocyte population was inversely correlated with the percentage of marker-positive cells (n = 11, P = .01), suggesting that the ability to induce MMP-9 may be compromised in clonally derived monocytes. The inducible levels of MMP-9 were also inversely correlated with marrow cellularity observed in biopsies from MDS patients (P < .001). We conclude that monocytes can express MMP-9 in response to stromal factors and that this response may be significantly decreased in MDS-derived monocytes.