Neutropenic Animals Research Articles

Neutrophils Mediate Pulmonary Artery Thrombosis In Situ.

Pulmonary embolism is a life-threatening condition, which can result in respiratory insufficiency and death. Blood clots occluding branches of the pulmonary artery (PA) are traditionally considered to originate from thrombi in deep veins (usually in legs). However, growing evidence suggests that occlusion of the vessels in the lungs can develop without preceding deep vein thrombosis (DVT). In this work, we used an inferior vena cava (IVC) complete ligation model of DVT in Wistar rats to explore the possibility and mechanisms of PA thrombosis under the conditions where all routes of thrombotic mass migration from peripheral veins are blocked. We demonstrate that rats both with normal and reduced neutrophil counts developed thrombi in the IVC, although, neutropenia caused a substantial decrease in thrombus size and a shift from fresh fibrin toward mature fibrin and connective tissue inside the thrombus. Massive fibrin deposition was found in the PA branches in the majority of DVT rats with normal neutrophil counts, but in none of the neutropenic animals. Neutrophil ablation also abolished macroscopic signs of lung damage. Altogether, the results demonstrate that thrombi in the lung vasculature can form in situ by mechanisms that require local neutrophil recruitment taking place in the DVT setting.

Neutrophil-suppressive activity over T-cell proliferation and fungal clearance in a murine model of Fonsecaea pedrosoi infection

Neutrophils are essential to control several fungal infections. These cells are commonly known for their pro-inflammatory activities. However, some studies have demonstrated the anti-inflammatory properties of neutrophils during certain infectious diseases, culminating in the inhibition of T cell proliferation. Chromoblastomycosis (CBM) is a deep and progressive mycosis that affects thousands of people worldwide. Although neutrophil infiltrates are observed in the lesion histopathology, the fungus can overtake the immune system response and destroy the host-infected tissue. The present study demonstrated that neutropenic animals had an increase in the IL-6 production in the spleen and liver, followed by a lower fungal burden in these organs up to 14 days of infection. Neutropenic animals also showed a lower F. pedrosoi-specific antibody production 14-days post infection and higher T-cell proliferation in the in vitro experiments after stimulation with F. pedrosoi-purified proteins. Taken together, our results suggest that the presence of regulatory neutrophils in the mouse model of F. pedrosoi infection could act favoring the spread of the fungus and the chronicity of the infection. These findings shed light on the CBM treatment, which might target neutrophil polarization as a new therapy approach to treat CBM lesions.

In Vivo Efficacy of Olorofim against Systemic Scedosporiosis and Lomentosporiosis.

Clinically relevant members of the Scedosporium/Pseudallescheria species complex and Lomentospora prolificans are generally resistant against currently available systemic antifungal agents in vitro, and infection due to these species is difficult to treat. We studied the in vivo efficacy of a new fungicidal agent, olorofim (formerly F901318), against scedosporiosis and lomentosporiosis in neutropenic animals. Cyclophosphamide-immunosuppressed CD-1 mice infected by Scedosporium apiospermum, Pseudallescheria boydii (Scedosporium boydii), and Lomentospora prolificans were treated by intraperitoneal administration of olorofim (15 mg/kg of body weight every 8 h for 9 days). The efficacy of olorofim treatment was assessed by the survival rate at 10 days postinfection, levels of serum (1-3)-β-d-glucan (BG), histopathology, and fungal burdens of kidneys 3 days postinfection. Olorofim therapy significantly improved survival compared to that of the untreated controls; 80%, 100%, and 100% of treated mice survived infection by Scedosporium apiospermum, Pseudallescheria boydii, and Lomentospora prolificans, respectively, while less than 20% of the control mice (phosphate-buffered saline [PBS] treated) survived at 10 days postinfection. In the olorofim-treated neutropenic CD-1 mice infected with any of the three species, serum BG levels were significantly suppressed and fungal DNA detected in the target organs was significantly lower than in controls. Furthermore, histopathology of kidneys revealed no or only a few lesions with hyphal elements in the olorofim-treated mice, while numerous fungal hyphae were present in control mice. These results indicate olorofim to be a promising therapeutic agent for systemic scedosporiosis/lomentosporiosis, devastating emerging fungal infections that are difficult to treat with currently available antifungals.

Biological depletion of neutrophils attenuates pro-inflammatory markers and the development of the psoriatic phenotype in a murine model of psoriasis

Although neutrophils are considered a histologic hallmark of psoriasis, their pathophysiologic role in psoriasis remains unclear. We characterized the effects of neutrophil depletion via injection of monoclonal antibody 1A8 on the development of imiquimod (IMQ)-induced psoriatic lesions in a murine model. Lesions were followed with photographs and histologic analysis, revealing reduced psoriasiform scale and epidermal hyperplasia in neutrophil-depleted. ELISA and flow cytometry were used to determine relative levels of cytokines and immune cells. Compared to controls, IMQ-treated neutropenic mice had significantly lower levels of macrophages in tissue samples (P < .05) and displayed significantly lower numbers of CD4+ T-cells (P < .05). Neutropenic animals exhibited lower levels of TNF-α, IFN-γ, and IL-1β than controls (P < .05). These results show that neutropenia reduces the development of psoriasiform skin lesions and substantially decreases infiltration of pro-inflammatory cytokines and immune cells to IMQ-induced cutaneous lesions, suggesting an active role of neutrophils in maintaining inflammation in psoriasis.

Neutrophils mediate early cerebral cortical hypoperfusion in a murine model of subarachnoid haemorrhage

Cerebral hypoperfusion in the first hours after subarachnoid haemorrhage (SAH) is a major determinant of poor neurological outcome. However, the underlying pathophysiology is only partly understood. Here we induced neutropenia in C57BL/6N mice by anti-Ly6G antibody injection, induced SAH by endovascular filament perforation, and analysed cerebral cortical perfusion with laser SPECKLE contrast imaging to investigate the role of neutrophils in mediating cerebral hypoperfusion during the first 24 h post-SAH. SAH induction significantly increased the intracranial pressure (ICP), and significantly reduced the cerebral perfusion pressure (CPP). At 3 h after SAH, ICP had returned to baseline and CPP was similar between SAH and sham mice. However, in SAH mice with normal neutrophil counts cortical hypoperfusion persisted. Conversely, despite similar CPP, cortical perfusion was significantly higher at 3 h after SAH in mice with neutropenia. The levels of 8-iso-prostaglandin-F2α in the subarachnoid haematoma increased significantly at 3 h after SAH in animals with normal neutrophil counts indicating oxidative stress, which was not the case in neutropenic SAH animals. These results suggest that neutrophils are important mediators of cortical hypoperfusion and oxidative stress early after SAH. Targeting neutrophil function and neutrophil-induced oxidative stress could be a promising new approach to mitigate cerebral hypoperfusion early after SAH.

CD11b Regulates Fungal Outgrowth but Not Neutrophil Recruitment in a Mouse Model of Invasive Pulmonary Aspergillosis

Background and Aims: In immunosuppressed individuals Aspergillus (A.) fumigatus is a frequent cause of invasive pulmonary aspergillosis (IPA) which is highly associated with relevant morbidity and mortality. Moreover, it often occurs in patients suffering from leukocyte-adhesion deficiency type 1 (LAD1) which is triggered by a functional loss of CD18 in ß2 integrin receptors as these receptors consist of an alpha subunit (CD11a-CD11d) and CD18 as the common beta subunit. ß2 integrin receptors are differentially expressed by leukocytes, and are required for cell-cell interaction, transendothelial migration, uptake of opsonized pathogens, and cell signaling processes. Here, we asked for the importance of CD11b/CD18 also termed MAC-1 which is required for phagocytosis of opsonized A. fumigatus conidia by polymorphonuclear neutrophils (PMN) for control of pulmonary A. fumigatus infection.Methods: We used a murine IPA model (C57BL/6) and challenged CD11b deficient (CD11b-/-) or wild type (WT) mice with A. fumigatus conidia intratracheally. Afterwards, some mice were sacrificed 24h after infection. In these mice PMN recruitment and cytokine patterns were examined by analyzing bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) using flow cytometry, cytospin analysis, and cytometric bead array. Additionally, pulmonary fungal clearance and inflammation in murine lungs were analyzed by fungal culture assays and histopathologic examination using a scoring system. Furthermore, survival was studied with neutropenic animals serving as positive controls. To determine PMNs phagocytic activity and fungal killing capacity ex vivo, PMN were purified from bone marrow of CD11b-/- or WT mice by magnetic cell sorting using Ly6G specific antibodies. Afterwards, isolated PMN were stimulated with pacific blue-labeled tomato red-fluorescent modified A. fumigatus conidia and analyzed by flow cytometry.Results: We found that lung homogenates from CD11b-/- mice obtained 24h after infection showed an enhanced fungal burden as compared to lungs from WT mice. In contrast, lung tissue from infected CD11b-/- mice displayed impaired pulmonary inflammation as assessed by Hematoxilin-Eosin (HE) staining. Furthermore, the number of mucus-producing cells in bronchi of A. fumigatus infected CD11b-/- mice was decreased compared to cells in bronchi of WT mice. However, we observed markedly higher numbers of PMN in BALF of infected CD11b-/- mice compared to corresponding WT mice samples. In contrast, BALF derived from infected CD11b-/- mice contained lower levels of three different proinflammatory cytokines (TNF-α, IL-1α, IL-1β) compared to BALF from WT mice while levels of other cytokines and chemokines (IL-5, IL-6, IL-10, GM-CSF, CXCL1, CCL2) were comparable. In contrast, we measured higher levels of the chemokine CCL5 known as a relevant chemoattractant in innate and adaptive immune cells in BALF obtained from CD11b-/- mice. Interestingly, numbers of PMN, lymphocytes, and monocytes in the peripheral blood of A. fumigatus infected mice did not differ in a genotype-dependent manner. However, CD11b-/- mice PMN showed lower phagocytic activity than WT PMN, indicating an impaired capability to clear A. fumigatus. Nevertheless, CD11b-/- mice were finally characterized by similar long-term survival as WT mice after IPA induction while the PMN-depleted control mice died as expected.Conclusions: Our study demonstrates, that CD11b deficiency on myeloid cells affects the early course of IPA. This may be due to the importance of MAC-1 for PMN effector functions, and their interplay with DC and other leukocytes. Further work is necessary to elucidate the long-term course of IPA in CD11b-/- mice with regard to the interplay of PMN with dendritic cells, the efficacy of adaptive immune responses, and the potential pulmonary overexpression of CCL5. Beyond patients with LAD1 syndrome, we suggest our findings as being clinically relevant in other immunocompromised patients suffering from severe opportunistic infections. DisclosuresRadsak:Celgene: Honoraria, Other: Travel grant; Jazz Pharmaceuticals: Other: Travel grant; TEVA: Consultancy; Gilead: Other: Travel grant; Takeda: Consultancy; Novartis: Consultancy, Honoraria; Daiichi Sankyo: Honoraria, Other: Travel grant.

Murine model of invasive pulmonary Aspergillosis: Follow-up of tissue injury, fungal burden and mortality with distinct elastase production strains

To study invasive pulmonary Aspergillosis (IPA), we depleted neutrophils in mice using the monoclonal antibody anti-Gr-1/Ly-6G. Immunocompetent and neutropenic mice were infected via intratracheal with conidia of Aspergillus fumigatus clinical isolates, characterized as either higher or lower elastase producers. Neutropenic animals exhibited 100% mortality in 5 days, for both strains, and were observed survival curves overlapped, lungs with angioinvasion, rupture of bronchial and vascular walls, associated with exuberance of conidia filamentation. The immunocompetent animals infected with the lower elastase producer strain presented with upregulated inflammatory processes, and a lack of conidia filamentation in the tissue. The fungal burden in the lungs was not different in the immunocompetent and neutropenic groups. These findings confirm the protective role of neutrophils against A. fumigatus and suggest that the fungal elastinolytic activity is not a critical virulence factor but may be involved in tissue injury.

Impaired lung repair during neutropenia can be reverted by matrix metalloproteinase-9

BackgroundNeutrophils may cause tissue disruption during migration and by releasing cytotoxic molecules. However, the benefits of neutrophil depletion observed in experimental models of lung injury do not correspond with the...

The Impact of NFAT Inhibition on Neutrophil Effector Functions in Patients after Allogeneic Hematopoietic Stem Cell Transplantation and on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporin Α Treated and NFATc1LysM Mice

Background and Aims: Immunosuppressive medication e.g. by calcineurin inhibitors substantially contributes to the risk for opportunistic fungal infections in patients after allogeneic transplantation (HSCT). It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin especially in T cells. Additionally, recent data in rodent models indicate that NFAT also seems to play a relevant role in innate antifungal immune responses by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation.Methods: Firstly, isolated PMN from healthy donors were analyzed in vitro in absence or presence of CsA regarding their effector functions and activation-induced release of inflammatory mediators. Consecutively, blood samples of CsA-treated patients after allogeneic HSCT (n=17) and healthy donors (n=8) were analyzed ex vivo at two different time points as described above. Secondly, we used a murine IPA model (C57BL/6) and treated mice with CsA (18 mg/kg/d) or vehicle and challenged them with Aspergillus fumigatus (A. f.) conidia intratracheally. PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) using flow cytometry and cytometric bead array and murine lungs by fungal culture assays and histopathologic examination. Furthermore, survival was studied with neutropenic animals serving as positive controls. Moreover, LysM-specific NFATc1 knockout (NFATc1LysM) mice were bred lacking NFATc1 expression solely in myelomonocytic cells. These animals were also infected with A. f. and analyzed as further mentioned. In addition, we investigated myelopoiesis and myeloid differentiation by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1LysMmice using flow cytometry and simultaneously counting PMN in PB under steady state conditions.Results: CsA enhanced phagocytosis of PMN in vitro and ex vivo in patients’ blood samples (54.2 % +/- 4.1 (patients) vs. 43.8 +/- 1.5, LPS, p=0.006). Moreover, PMNs migratory capabilities were reduced in vitro, whereas other effector functions or release of IL-8 were rather unaffected. PMNs of CsA-treated patients showed increased activation, degranulation and production of inflammatory mediators, but production of ROS was slightly decreased. In our in vivo model, IPA was lethal in neutropenic mice whereas solely CsA or vehicle treated mice survived the infection. CsA treatment resulted in enhanced PMN recruitment in BAL by trend, while pulmonary inflammation and PMN counts in PB remained stable. Indeed, fungal clearance was clearly constrained in CsA treated animals (2.1 x 105 CFU/lung +/- 0.5 (CsA) vs. 1.7 x 105 +/- 0.2, p<0.005). In our murine knockout model, NFATc1LysM mice infected with A. f. showed unimpaired survival without displaying detectable differences in PMN recruitment or fungal clearance. However, pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice. Interestingly, BALs of CsA treated mice showed increased levels of IL-6 by trend (4634 pg/mL +/- 1073 (CsA) vs. 3108 +/- 729, p=0.48) but decreased levels of MCP-1 and TNF-α. In contrast, MCP-1, RANTES and TNF-α were enhanced by trend in BALs of NFATc1LysM mice, while IL-6 was reduced compared to wild type controls (3762 pg/mL +/- 729 vs. 4770 +/- 1613, p=0.81). PMN counts in PB were unaffected in NFATc1LysM mice but distribution of bone marrow derived murine myeloid progenitor cells was clearly impaired especially in megakaryocyte-erythroid progenitor cells (1.2 x 105 cells +/- 0.2 (NFATc1LysM) vs. 2.7 +/- 0.6, p=0.015), whereas solely CsA treatment had no influence.Conclusion: Results from our in vitro and ex vivo studies on patients' blood samples as well as from our murine in vivo IPA model indicate that NFAT regulates not only myelopoiesis, but also PMN functionalities in mice and humans. Nevertheless, these interactions are obviously multidimensional and potentially derive from involvement of different pathways. The underlying molecular mechanisms and clinical relevance of our findings in HSCT remain to be determined. DisclosuresNo relevant conflicts of interest to declare.

Cryopreserved Ex Vivo-Expanded Allogeneic Myeloid Progenitor Cell Product Protects Neutropenic Mice From a Lethal Fungal Infection.

Severe neutropenia induced by chemotherapy or conditioning for hematopoietic cell transplantation often results in morbidity and mortality due to infection by opportunistic pathogens. A system has been developed to generate ex vivo-expanded mouse myeloid progenitor cells (mMPCs) that produce functional neutrophils in vivo upon transplantation in a pathogen challenge model. It has previously been demonstrated that transplantation of large numbers of freshly isolated myeloid progenitors from a single donor provides survival benefit in radiation-induced neutropenic mice. In the present work, an ex vivo-expanded and cryopreserved mMPC product generated from an allogeneic donor pool retains protective activity in vivo in a lethal fungal infection model. Infusion of the allogeneic pooled mMPC product is effective in preventing death from invasive Aspergillus fumigatus in neutropenic animals, and protection is dose dependent. Cell progeny from the mMPC product is detected in the bone marrow, spleen, blood, and liver by flow cytometry 1 week postinfusion but is no longer evident in most animals 4 weeks posttransplant. In this model, the ex vivo-generated pooled allogeneic mMPC product (i) expands and differentiates in vivo; (ii) is functional and prevents death from invasive fungal infection; and (iii) does not permanently engraft or cause allosensitization. These data suggest that an analogous ex vivo-expanded human myeloid progenitor cell product may be an effective off-the-shelf bridging therapy for the infectious complications that develop during hematopoietic recovery following hematopoietic cell transplantation or intensive chemotherapy.

The Impact of NFAT Inhibition on Neutrophil Antifungal Defense and Myelopoiesis in Cyclosporine A Treated and NFATc1LysM Mice

Background and Aims: Immunodeficient patients after allogeneic stem cell transplantation (HSCT) are heavily threatened by opportunistic fungal infections like invasive pulmonary aspergillosis (IPA), partly due to immunosuppressive medication e.g. by calcineurin inhibitors like cyclosporine A (CsA) or tacrolimus. It is well known that the nuclear factor of activated T cells (NFAT) is an important transcription factor downstream of calcineurin in the adaptive immune system especially in T cells. Additionally, there is a growing body of evidence that NFAT also plays a substantial role in innate immune response against invasive fungal diseases by polymorphonuclear neutrophils (PMN), as well as in regulation of myelopoiesis and myeloid differentiation, as indicated by recent data in rodent models.Methods: Firstly, we used a murine IPA model (C57BL/6) to clarify the role of NFAT in antifungal innate immune response in vivo. To do so, we treated mice intraperitoneally with CsA (18mg/kg/d) or vehicle for 2 weeks and challenged them with Aspergillus fumigatus (A. f.) conidia intratracheally. 24 hours later, some mice were sacrificed and PMN recruitment to the lungs and pulmonary fungal clearance were examined by analyzing bronchoalveolar lavages (BAL) and peripheral blood (PB) by flow cytometry and murine lungs by fungal culture assays and histopathologic examination. In addition, survival of remaining infected mice was studied with neutropenic animals (by depletion with anti-Gr1) serving as positive controls. Secondly, LysM-specific NFATc1 knockout (NFATc1LysM) mice were bred lacking NFATc1 expression solely in myelomonocytic cells (i.e. PMN and monocytes). Furthermore, these animals were infected with A. f. and analyzed as described above. Secondly, we investigated myelopoiesis and myeloid differentiation under steady state conditions by quantifying bone marrow derived myeloid progenitor cells from CsA treated or NFATc1LysM mice using flow cytometry and simultaneously counting PMN in PB.Results: While the infection was lethal in CsA or vehicle treated neutropenic mice, all CsA or vehicle treated mice survived the infection. CsA treated mice showed enhanced PMN recruitment in BAL by trend (55.2% +/- 12.0 (CsA) vs. 33.7% +/- 8.0 (control), mean +/- SEM, p=0.053), whereas pulmonary inflammation and PMN counts in PB were comparable to controls. In contrast, fungal clearance was clearly impaired in animals after CsA treatment (2.1 x 105 CFU/lung after 48 hours +/- 0.5 (CsA) vs. 1.7 x 105 +/- 0.2 (control), p<0.005). Along with that, NFATc1LysM mice infected with A. f. showed unimpaired survival. However, there were no detectable differences in PMN recruitment or fungal clearance, whereas pulmonary inflammation and PMN counts in PB seemed to be more pronounced in knockout mice (1.0 inflammation points/lung +/- 0.12 (NFATc1LysM) vs. 0.7 +/- 0.07 (control), p=0.057; 1.5 x 103 PMN/µl +/- 0.2 (NFATc1LysM) vs. 0.9 +/- 0.1 (control), p=0.036). Distribution of bone marrow derived murine myeloid progenitor cells was unaffected through NFAT inhibition by CsA but clearly impaired in NFATc1LysM mice especially in megakaryocyte-erythroid progenitor cells (1.2 x 105 cells +/- 0.2 (NFATc1LysM) vs. 2.7 +/- 0.6, p=0.015) whereas PMN blood counts in PB were unchanged.Conclusions: In a mouse model, NFAT inhibition via treatment with CsA does not influence survival after infection with A. f. in vivo but affects PMN recruitment and local fungal clearance. To some extent this may be due to impaired PMN phagocytic and migratory capabilities as indicated by our in vitro and ex vivo studies (data not shown). However, solely NFATc1 downregulation in PMN apparently results in slightly different effects given that infected NFATc1LysM mice displayed enhanced pulmonary inflammation and elevated PMN blood counts compared to controls. Additionally, NFATc1 inhibition in NFATc1LysM mice leads to constrained myelopoiesis under steady state conditions without affecting peripheral PMN blood counts compared to untreated wild type controls. Further studies are needed to clarify underlying mechanisms and clinical relevance in HSCT of our findings. DisclosuresNo relevant conflicts of interest to declare.

Single or 2-Dose Micafungin Regimen for Treatment of Invasive Candidiasis: Therapia Sterilisans Magna!

The time the earth takes to rotate its axis (the day) has dictated how often pharmaceutical compounds are dosed. The scientific link between the 2 events is materia medica arcana. As an example, in the treatment of invasive candidiasis, antifungal therapy with intravenous micafungin is dosed daily. A literature review revealed population pharmacokinetic analyses, in vivo pharmacokinetics/pharmacodynamics studies, and maximum-tolerated-dose studies of micafungin that examined optimal micafungin dosing strategies. The half-life of micafungin in patient blood was 14 hours in several studies, but was even longer in different organs, so that the concentration will persist above minimum inhibitory concentrations of Candida species for several days. Studies in mice and rabbits with persistent neutropenia and disseminated candidiasis, otherwise fatal, demonstrated that a single large dose of micafungin could clear disseminated candidiasis, even though the micafungin half-life in such animals is shorter than in humans. Human pharmacokinetics/pharmacodynamics studies confirmed this link between micafungin efficacy and the ratio of the area under the concentration-time curve, and the optimal exposures initially identified in neutropenic animals. Maximum tolerated dose studies have demonstrated safety of 900 mg administered daily for several weeks, whereas case reports demonstrate efficacy and safety of single 1400-mg doses. Thus, a single dose of micafungin, or 2 such doses within a few days of each other, is not only logical, but might even lead to faster clearance of Candida.

The novel immunotherapeutic oligodeoxynucleotide IMT504 protects neutropenic animals from fatal Pseudomonas aeruginosa bacteremia and sepsis.

IMT504 is a novel immunomodulatory oligonucleotide that has shown immunotherapeutic properties in early preclinical and clinical studies. IMT504 was tested in a neutropenic rat model of Pseudomonas aeruginosa bacteremia and sepsis. This animal system recapitulates many of the pathological processes found in neutropenic patients with Gram-negative, bacterial infections. The research was conducted in the setting of an academic research laboratory. The test subjects were Sprague-Dawley rats. Animals were rendered neutropenic by administration of cyclophosphamide, colonized with P. aeruginosa by oral feeding, and then randomized to receive IMT504 over a range of doses and treatment regimens representing early and late therapeutic interventions. IMT504 immunotherapy conferred a significant survival advantage over the 12-day study period compared with the results seen with placebo-treated animals when the therapy was administered at the onset of neutropenia and even in the absence of antibiotics and after the onset of fever and systemic infection. Notably, even late salvage IMT504 monotherapy was highly effective (13/14 surviving rats with IMT504 therapy versus 2/14 controls, P=<0.001). Moreover, late salvage IMT504 monotherapy was as effective as antibiotic therapy (13/14 surviving rats versus 21/21 rats, P=0.88). In addition, no antagonism or loss of therapeutic efficacy was noted with combination therapy of IMT504 plus antibiotics. IMT504 immunotherapy provides a remarkable survival advantage in bacteremia and sepsis in neutropenic animals and deserves further study as a new treatment option in patients with, or at risk for, severe Gram-negative bacterial infections and sepsis.

Pegylated feline granulocyte colony-stimulating factor increases neutrophil levels in cats

Neutropenia can often be corrected by treatment with granulocyte-colony stimulating factor (G-CSF) and off-label use of commercial human G-CSF (HuG-CSF) is a commonly used treatment for neutropenic animals. However, long-term HuG-CSF treatment can be associated with adverse effects, including neutropenia. Here, feline (Fe) G-CSF was produced in Pichia pastoris, pegylated (Peg) FeG-CSF and tested in cats. A randomized controlled clinical trial was conducted to evaluate the efficacy of PegFeG-CSF compared to FeG-CSF or HuG-CSF in FIV-infected (n=14), FIV-uninfected healthy cats (n=19), and in HuG-CSF-induced neutropenic cats (n=4).Daily FeG-CSF doses induced higher neutrophil production than HuG-CSF after the second week of treatment (P⩽0.002). Weekly doses of PegFeG-CSF induced higher neutrophil counts and showed greater sustained activity than weekly doses of FeG-CSF. PegFeG-CSF provided the most therapeutic and sustainable neutrophil production (P<0.001) in both FIV-uninfected and FIV-infected cats, without the development of neutralizing antibodies. Conversely, all HuG-CSF-treated cats developed neutralizing antibodies, suggesting cross-reactive antibodies to endogenous G-CSF in a majority of the cases with severe neutropenia. Strikingly, when PegFeG-CSF was used to rescue cats with HuG-CSF-induced neutropenia, clinically normal neutrophil numbers returned. Thus, PegFeG-CSF appears to be a superior treatment for neutropenia in feline patients.

Use of Pharmacokinetic/Pharmacodynamic Systems Analyses to Inform Dose Selection of Tedizolid Phosphate

In the Staphylococcus aureus neutropenic murine thigh-infection model, the ratio of the free area under the 24-hour concentration-time curve to the minimum inhibitory concentration (fAUC/MIC) was found to be the pharmacodynamic index most closely linked to bacterial effect, with a ratio of approximately 50 producing a static effect. Further work was undertaken in neutropenic versus non-neutropenic animals. The presence of granulocytes increased the activity of tedizolid considerably, 25-fold on average, and maximal effect was achieved at an exposure equivalent to approximately 200 mg tedizolid phosphate per day in humans (dosing regimen used in phase 2 and 3 clinical trials). The fAUC/MIC was also found to be the pharmacodynamically linked variable in the S. aureus neutropenic murine pneumonia model; the fAUC/MIC ratio required for a static effect was approximately 20. Pharmacokinetic (PK) data demonstrate that tedizolid penetrates well into the epithelial lining fluid (ELF) of the lung. Data from the pneumonia infection model and ELF penetration PK study support exploring its use in pneumonia.

Intraperitoneal prophylaxis with CpG oligodeoxynucleotides protects neutropenic mice against intracerebral Escherichia coli K1 infection

BackgroundProphylaxis with unmethylated cytosine phosphate guanidine (CpG) oligodeoxynucleotides (ODN) protects against several systemic experimental infections. Escherichia coli is a major cause of Gram-negative neonatal bacterial meningitis and also causes meningitis and meningoencephalitis in older and immunocompromised patients.MethodsWild-type (wt) and Toll-like receptor 9 (TLR9)-deficient mice were rendered neutropenic by intraperitoneal administration of the anti-Ly-6G monoclonal antibody. Immunocompetent and neutropenic mice received intraperitoneal CpG ODN or vehicle 72 h prior to induction of E. coli K1 meningoencephalitis.ResultsPre-treatment with CpG ODN significantly increased survival of neutropenic wt mice from 33% to 75% (P = 0.0003) but did not protect neutropenic TLR9-/- mice. The protective effect of CpG ODN was associated with an enhanced production of interleukin (IL)-12/IL-23p40 with sustained increased levels in serum and spleen at least for 17 days after conditioning compared to buffer-treated animals. CpG-treated neutropenic wt mice showed reduced bacterial concentrations and increased recruitment of Ly6ChighCCR2+ monocytes in brain and spleen 42 h after infection. The levels of macrophage inflammatory protein 1α (MIP-1α) and interferon gamma (IFN-γ) in spleen were higher 42 h after infection in CpG-treated compared to buffer-treated neutropenic animals. In immunocompetent mice, prophylaxis with CpG ODN did not significantly increase survival compared to the buffer group (60% vs. 45%, P = 0.2).ConclusionsThese findings suggest that systemic administration of CpG ODN may help to prevent bacterial CNS infections in immunocompromised individuals.

KPC-Producing, Multidrug-Resistant Klebsiella pneumoniae Sequence Type 258 as a Typical Opportunistic Pathogen

The virulence of a KPC-producing Klebsiella pneumoniae sequence type 258 (ST258) strain representing those circulating in Greece was assessed in a mouse septicemia model. The strain was virtually avirulent (50% lethal dose, >10(8) and 5 × 10(7) CFU for immunocompetent and neutropenic animals, respectively). Also, it was highly susceptible to serum killing, rapidly phagocytosed in vitro, and classified as K41, which is not among the virulent capsular types. The findings indirectly support the notion that high ST258-associated mortality is largely due to inefficient antimicrobial treatment.

Analysis of theStaphylococcus aureusabscess proteome identifies antimicrobial host proteins and bacterial stress responses at the host-pathogen interface

Abscesses are a hallmark of invasive staphylococcal infections and the site of a dynamic struggle between pathogen and host. However, the precise host and bacterial factors that contribute to abscess formation and maintenance have not been completely described. In this work, we define the Staphylococcus aureus abscess proteome from both wild-type and neutropenic mice to elucidate the host response to staphylococcal infection and uncover novel S. aureus virulence factors. Among the proteins identified, the mouse protein histone H4 was enriched in the abscesses of wild-type compared with neutropenic animals. Histone H4 inhibits staphylococcal growth in vitro demonstrating a role for this protein in the innate immune response to staphylococcal infection. These analyses also identified staphylococcal proteins within the abscess, including known virulence factors and proteins with previously unrecognized roles in pathogenesis. Within the latter group was the universal stress protein Usp2, which was enriched in kidney lesions from neutropenic mice and required for the S. aureus response to stringent stress. Taken together, these data describe the S. aureus abscess proteome and lay the foundation for the identification of contributors to innate immunity and bacterial pathogenesis.

Promotion of Colonization and Virulence by Cholera Toxin Is Dependent on Neutrophils

The innate immune response to Vibrio cholerae infection is poorly understood, but this knowledge is critical for the design of safe, effective vaccines. Using an adult mouse intestinal infection model, this study examines the contribution of neutrophils to host immunity, as well as the effect of cholera toxin and other secreted factors on this response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to similar survival rates of mice infected with low or moderate doses of toxigenic V. cholerae El Tor O1. At a high dose, neutropenic mice showed increased rates of survival compared to neutrophil-replete animals. Expression of cholera toxin was found to be protective to the neutropenic host, and this phenotype can be replicated by the administration of purified toxin. Neutrophils do not effectively clear colonizing bacteria from the small intestine, nor do they alter induction of early immune-modulating signals. In both neutropenic and neutrophil-replete animals, the local response to infection is characterized by expression of interleukin 6 (IL-6), IL-10, and macrophage inflammatory protein 2 alpha (MIP-2). Overall, these data indicate that the innate immune response to toxigenic V. cholerae infection differs dramatically from the host response to nontoxigenic infection or vaccination, where neutrophils are protective to the host. In the absence of neutrophils, cholera toxin induces immunomodulatory effects that increase host survival. In cholera toxin-producing strains, similar to nontoxigenic infection, accessory toxins are critical to virulence, indicating that cholera toxin and the other secreted toxins modulate the host response by different mechanisms, with both contributing to bacterial persistence and virulence.

Pegfilgrastim Administered in an Abbreviated Schedule, Significantly Improved Neutrophil Recovery after High-Dose Radiation-Induced Myelosuppression in Rhesus Macaques

Conventional daily administration of filgrastim is effective in reducing the duration of severe neutropenia and enhancing survival following lethal radiation, myelosuppressive cytotoxic therapy or myeloablation and stem cell transplantation. A sustained-duration form of filgrastim, pegfilgrastim has significantly simplified scheduling protocols after chemotherapy-induced neutropenia to a single injection while maintaining the therapeutic effectiveness of daily administration of filgrastim. We examined the ability of a single or double (weekly) administration of pegfilgrastim to significantly improve neutrophil recovery in a rhesus macaque model of severe radiation-induced myelosuppression. Animals were exposed to potentially lethal 6 Gy total-body X radiation. After irradiation all animals received supportive care and were administered either pegfilgrastim at 300 μg/kg on day 1 or day 1 and day 7 post exposure, or filgrastim at 10 μg/kg/day initiated on day 1 post exposure and continued daily through neutrophil recovery. Pharmacokinetic parameters and neutrophil-related values for duration of neutropenia, neutrophil nadir, time to recovery to an absolute neutrophil count ≥500/μL or ≥2000/μL, and days of antibiotic support were determined. Effective plasma concentrations of pegfilgrastim were maintained in neutropenic animals until after the onset of hematopoietic recovery, which is consistent with neutrophil-dependent properties of elimination. Administration of pegfilgrastim at day 1 and day 7 was most effective at improving neutrophil recovery compared to daily administration of filgrastim or a single injection of pegfilgrastim on day 1, after severe, radiation-induced myelosuppression in rhesus macaques.