Adverse immunostimulation caused by impurities: The dark side of biopharmaceuticals.

Drug safety is an important issue, especially in the experimental phases of development. Adverse immunostimulation (AI) is sometimes encountered following treatment with biopharmaceuticals, which can be life-threatening if it results in a severe systemic inflammatory reaction. Biopharmaceuticals that unexpectedly induce an inflammatory response still enter the clinic, even while meeting all regulatory requirements. Impurities (of microbial origin) in biopharmaceuticals are an often-overlooked cause of AI. This demonstrates that the current guidelines for quality control and safety pharmacology testing are not flawless. Here, based on two case examples, several shortcomings of the guidelines are discussed. The most important of these are the lack of sensitivity for impurities, lack of testing for pyrogens other than endotoxin, and the use of insensitive animal species and biomarkers in preclinical investigations. Moreover, testing for the immunotoxicity of biopharmaceuticals is explicitly not recommended by the international guidelines. Publication of cases of AI is pivotal, both to increase awareness and to facilitate scientific discussions on how to prevent AI in the future.

Establishing the Production of Male Schistosoma mansoni Cercariae for a Controlled Human Infection Model.

To accelerate the development of novel vaccines for schistosomiasis, we set out to develop a human model for Schistosoma mansoni infection in healthy volunteers. During natural infections, female schistosomes produce eggs that give rise to morbidity. Therefore, we produced single-sex, male Schistosoma mansoni cercariae for human infection without egg production and associated pathology. Cercariae were produced in their intermediate snail hosts in accordance with the principles of good manufacturing practice (GMP). The application of GMP principles to an unconventional production process is a showcase for the controlled production of complex live challenge material in the European Union or under Food and Drug Administration guidance.

Generation of mesenchymal stem cells as a medicinal product in organ transplantation.

PURPOSE OF REVIEW: Mesenchymal stem cells (MSCs) are emerging as an alternative treatment in solid-organ transplantation. The use of MSCs as a therapeutic product requires the translation of basic research protocols into a production process under good manufacturing practice (GMP) to obtain a safe product of high quality. This requires a different mindset from the academic setting of changing protocols into a well defined, controlled and documented process. This review describes some of the challenges faced by culturing MSCs as a medicinal product. RECENT FINDINGS: Clinical-grade MSCs are used in the clinical trials and proved to be safe as a medicinal product. Because of the differences in the type of MSCs and in the production process, clinical outcome is not always comparable. New standardized methods in the culture condition such as the use of alternatives for fetal bovine serum (FBS), standardized plating densities or the use of bioreactors may further standardize the production process. SUMMARY: To generate MSCs as a medicinal product in organ transplantation, regulation requires that MSCs have to be generated under GMP. During the whole production process, all critical steps should be known and described. Further steps should be taken to optimize and standardize the production process.

The microtubule regulator stathmin is an endogenous protein agonist for TLR3.

TLR3 recognizes dsRNAs and is considered of key importance to antiviral host-defense responses. TLR3 also triggers neuroprotective responses in astrocytes and controls the growth of axons and neuronal progenitor cells, suggesting additional roles for TLR3-mediated signaling in the CNS. This prompted us to search for alternative, CNS-borne protein agonists for TLR3. A genome-scale functional screening of a transcript library from brain tumors revealed that the microtubule regulator stathmin is an activator of TLR3-dependent signaling in astrocytes, inducing the same set of neuroprotective factors as the known TLR3 agonist polyinosinic:polycytidylic acid. This activity of stathmin crucially depends on a long, negatively charged alpha helix in the protein. Colocalization of stathmin with TLR3 on astrocytes, microglia, and neurons in multiple sclerosis-affected human brain indicates that as an endogenous TLR3 agonist, stathmin may fulfill previously unsuspected regulatory roles during inflammation and repair in the adult CNS.

Paramedic contact to balloon in less than 90 minutes: a successful strategy for st-segment elevation myocardial infarction bypass to primary percutaneous coronary intervention in a canadian emergency medical system.

INTRODUCTION: Few systems worldwide have achieved the benchmark time of less than 90 minutes from emergency medical services (EMS) contact to balloon inflation (E2B) for patients sustaining ST-segment elevation myocardial infarction (STEMI). We describe a successful EMS systems approach using a combination of paramedic and 12-lead electrocardiogram (ECG) software interpretation to activate a STEMI bypass protocol. OBJECTIVES: To determine the proportion of patients who met the benchmark of E2B in less than 90 minutes after institution of a regional paramedic activated STEMI bypass to primary PCI protocol. METHODS: We conducted a before-and-after observational cohort study over a 24-month period ending December 31, 2009. Included were all patients diagnosed with STEMI by paramedics trained in ECG acquisition and interpretation and transported by EMS. In the "before" phase of the study, paramedics gave emergency departments (EDs) advance notification of the arrival of STEMI patients and took the patients to the ED of the PCI center. In the "after" phase of the study, paramedics activated a STEMI bypass protocol in which STEMI patients were transported directly to the PCI suite, bypassing the local hospital EDs. Transmission of ECGs did not occur in either phase of the study. RESULTS: We compared the times for 95 STEMI patients in the before phase with the times for 80 STEMI patients in the after phase. The proportion for whom E2B was less than 90 minutes increased from 28.4% before to 91.3% after (p < 0.001). Median E2B time decreased from 107 minutes (interquartile range [IQR] = 30) before to 70 minutes (IQR = 24) after. Median D2B time decreased from 83 minutes (IQR = 34) before to 35 minutes (IQR = 19) after. Median E2D time increased from 21 minutes (IQR = 8) before to 32 minutes (IQR = 17) after. Median differences between phases were significant at p < 0.001. The rate of false-positive PCI laboratory activation during the after phase of the study was 12.4%. CONCLUSIONS: The proportion of patients with E2B times less than 90 minutes significantly improved through the implementation of a paramedic-activated STEMI bypass protocol. Further study is required to determine whether these benefits are reproducible in other EMS systems.

Field Implementation of Remote Ischemic Conditioning in ST-Segment-Elevation Myocardial Infarction: The FIRST Study.

BACKGROUND: Remote ischemic conditioning (RIC) is a noninvasive therapeutic strategy that uses brief cycles of blood pressure cuff inflation and deflation to protect the myocardium against ischemia-reperfusion injury. We sought to compare major adverse cardiovascular events (MACE) for patients who received RIC before PCI for ST-segment-elevation myocardial infarction (STEMI) compared with standard care. METHODS: We conducted a pre- and postimplementation study. In the preimplementation phase, STEMI patients were taken directly to the PCI lab. After implementation, STEMI patients received 4 cycles of RIC by paramedics or emergency department staff before PCI. The primary outcome was MACE at 90 days. Secondary outcomes included MACE at 30, 60, and 180 days. Inverse probability of treatment weighting using propensity scores estimated causal effects independent from baseline covariables. RESULTS: A total of 1667 (866 preimplementation, 801 postimplementation) patients were included. In the preimplementation phase, 13.4% had MACE at 90 days compared with 11.8% in the postimplementation phase (odds ratio [OR] 0.86, 95% CI 0.62-1.21). There were no significant differences in MACE at 30, 60, and 180 days. Patients presenting with cardiogenic shock or cardiac arrest before PCI were less likely to have MACE at 90 days (42.7% pre vs 27.8% post) if they received RIC before PCI (OR 0.52, 95% CI 0.27-0.98). CONCLUSIONS: A strategy of RIC before PCI for STEMI did not reduce 90-day MACE. Future research should explore the impact of RIC before PCI for longer-term clinical outcomes and for patients presenting with cardiogenic shock or cardiac arrest.

Inter-rater reliability and comfort in the application of a basic life support termination of resuscitation clinical prediction rule for out of hospital cardiac arrest.

STUDY OBJECTIVE: This study evaluates inter-rater reliability and comfort of BLS providers with the application of an out-of-hospital Basic Life Support Termination of Resuscitation (BLS TOR) clinical prediction rule. This rule suggests that continued BLS cardiac resuscitation is futile and can be terminated in the field if the following three conditions are met: (1) no return of spontaneous circulation; (2) no shock given prior to transport; (3) cardiac arrest not witnessed by EMS personnel. METHODS: Providers hypothetically applied the rule and rated their comfort level on a five-point Likert-type scale, from "very comfortable" to "very uncomfortable" during the prospective validation of a BLS TOR clinical prediction rule in out-of-hospital cardiac arrest conducted in 12 rural and urban communities [Morrison LJ, Visentin LM, Kiss A, et al. Validation of a rule for termination of resuscitation in out-of-hospital cardiac arrest. New Engl J Med 2006;355(5):478-87]. A Kappa score measured agreement between providers and compared to the correct interpretation of the rule. RESULTS: We compared mean comfort levels of providers who interpreted the rule correctly versus incorrectly. Of 1240 enrolled cases, 1184 (95.5%) had paramedic attendant forms and 1211 (97.7%) had driver forms and 1175 (94.7%) had both. Kappa for interpretation agreement between driver and attendant was 0.90 (95% CI, 0.87-0.92); between attendant and correct interpretation of the BLS TOR clinical prediction rule, 0.88 (95% CI, 0.85-0.91); between driver and correct interpretation of the BLS TOR clinical prediction rule, 0.88 (95% CI, 0.85-0.91). For instances in which both providers applied the rule correctly (607/635 [95.6%]), the providers were significantly more comfortable (chi(2)(4)=30.5, p<0.0001) than those instances in which they did not (28/635 [4.4%]. CONCLUSIONS: The vast majority of providers were able to apply the BLS TOR clinical prediction rule correctly and were comfortable doing so. This suggests that both reliability and comfort will remain high during routine application of the rule when paramedics are well trained as users of the rule.

Oral flavonoids delay recovery from experimental autoimmune encephalomyelitis in SJL mice.

Flavonoids are food components that appear to have potential beneficial health effects. There is a range of in vitro studies supporting the anti-oxidant and anti-inflammatory properties of flavonoids. Previously, we demonstrated that in vitro flavonoids, including luteolin and apigenin, inhibit proliferation and IFN-gamma production by murine and human autoimmune T cells. In the present study, we examined the effects of oral flavonoids as well as of curcumin on autoimmune T cell reactivity in mice and on the course of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. Continuous oral administration of flavonoids significantly affected antigen-specific proliferation and IFN-gamma production by lymph node-derived T cells following immunization with an EAE-inducing peptide. Both luteolin and apigenin suppress proliferative responses as they did in vitro, whereas IFN-gamma production on the other hand was enhanced. Other flavonoids exerted differential effects on proliferation and IFN-gamma production. The effects of flavonoids and curcumin on EAE were assessed using either passive transfer of autoimmune T cells or active disease induction. In passive EAE, flavonoids led to delayed recovery of clinical symptoms rather than to any reduction in disease. In active EAE, the effects were less pronounced but also, in this case, the flavonoid hesperitin delayed recovery. Oral curcumin had overall mild but beneficial effects. Our results indicate that oral flavonoids fail to beneficially influence the course of EAE in mice but, instead, suppress recovery from acute inflammatory damage.

Therapeutic Intervention in Multiple Sclerosis with Alpha B-Crystallin: A Randomized Controlled Phase IIa Trial.

UNLABELLED: As a molecular chaperone and activator of Toll-like receptor 2-mediated protective responses by microglia and macrophages, the small heat shock protein alpha B-crystallin (HspB5) exerts therapeutic effects in different animal models for neuroinflammation, including the model for multiple sclerosis (MS). Yet, HspB5 can also stimulate human antigen-specific memory T cells to release IFN-γ, a cytokine with well-documented detrimental effects during MS. In this study, we explored in a Phase IIa randomized clinical trial the therapeutic application of HspB5 in relapsing-remitting MS (RR-MS), using intravenous doses sufficient to support its protective effects, but too low to trigger pathogenic memory T-cell responses. These sub-immunogenic doses were selected based on in vitro analysis of the dose-response profile of human T cells and macrophages to HspB5, and on the immunological effects of HspB5 in healthy humans as established in a preparatory Phase I study. In a 48-week randomized, placebo-controlled, double-blind Phase IIa trial, three bimonthly intravenous injections of 7.5, 12.5 or 17.5 mg HspB5 were found to be safe and well tolerated in RR-MS patients. While predefined clinical endpoints did not differ significantly between the relatively small groups of MS patients treated with either HspB5 or placebo, repeated administration especially of the lower doses of HspB5 led to a progressive decline in MS lesion activity as monitored by magnetic resonance imaging (MRI), which was not seen in the placebo group. Exploratory linear regression analysis revealed this decline to be significant in the combined group receiving either of the two lower doses, and to result in a 76% reduction in both number and total volumes of active MRI lesions at 9 months into the study. These data provide the first indication for clinical benefit resulting from intervention in RR-MS with HspB5. TRIAL REGISTRATION: ClinicalTrials.gov Phase I: NCT02442557; Phase IIa: NCT02442570.

A novel rationale for inhibition of gelatinase B in multiple sclerosis: MMP-9 destroys alpha B-crystallin and generates a promiscuous T cell epitope.

The small heat shock protein alphaB-crystallin is considered as a candidate autoantigen in multiple sclerosis (MS) lesions. Gelatinase B or matrix metalloproteinase (MMP)-9 is a proteinase establishing various disease-promoting feedback loops in autoimmune diseases. Human alphaB-crystallin was digested with natural gelatinase B and all cleavage sites were identified by a combined approach of mass spectrometry and peptide sequencing analysis. Previously identified immunodominant and cryptic epitopes of alphaB-crystallin in mice and rats were generated and largely left intact by MMP-9 processing. The alphaB-crystallin peptide 1-16, generated as a remnant epitope, provoked a significant T cell response in alphaB-crystallin knockout mice. None of the remnant peptides was encephalitogenic when injected intracerebrally into mice or induced MMP-9 in vitro. Gelatinase B is thus able to release T cell epitopes from intact alphaB-crystallin, but their pathogenic role remains unclear.

The flavones luteolin and apigenin inhibit in vitro antigen-specific proliferation and interferon-gamma production by murine and human autoimmune T cells.

Plant-derived flavonoids are inhibitors of various intracellular processes, notably phosphorylation pathways, and potential inhibitors of cellular autoimmunity. In this study, the inhibiting effects of various flavonoids on antigen-specific proliferation and interferon-gamma (IFN-gamma) production by human and murine autoreactive T cells were evaluated in vitro. T-cell responses were evaluated for the human autoantigen alpha B-crystallin, a candidate autoantigen in multiple sclerosis, and for the murine encephalitogen proteolipid protein peptide PLP (139-151). The flavones apigenin and luteolin were found to be strong inhibitors of both murine and human T-cell responses while fisitin, quercitin, morin and hesperitin, members of the subclasses of flavonoles and flavanones, were ineffective. Antigen-specific IFN-gamma production was reduced more effectively by flavones than T-cell proliferation, suggesting that the intracellular pathway for IFN-gamma production in T cells is particularly sensitive to flavone inhibition. These results indicate that flavones but not flavanoles or flavanones are effective inhibitors of the potentially pathogenic function of autoreactive T cells. The effects of flavones were the same for human and murine autoreactive T cells, stressing the usefulness of animal models of autoimmunity for further studies on the effects of flavonones on autoimmune diseases.

Half-a-million strong: the emergency medical services response to a single-day, mass-gathering event.

INTRODUCTION: Emergency medical services (EMS) responses to mass gatherings have been described frequently, but there are few reports describing the response to a single-day gathering of large magnitude. OBJECTIVE: This report describes the EMS response to the largest single-day, ticketed concert held in North America: the 2003 "Toronto Rocks!" Rolling Stones Concert. METHODS: Medical care was provided by paramedics, physicians, and nurses. Care sites included ambulances, medically equipped, all-terrain vehicles, bicycle paramedic units, first-aid tents, and a 124-bed medical facility that included a field hospital and a rehydration unit. Records from the first-aid tents, ambulances, paramedic teams, and rehydration unit were obtained. Data abstracted included patient demographics, chief complaint, time of incident, treatment, and disposition. RESULTS: More than 450,000 people attended the concert and 1,870 sought medical care (42/10,000 attendees). No record was kept for the 665 attendees simply requesting water, sunscreen, or bandages. Of the remaining 1,205 patients, the average of the ages was 28 +/- 11 years, and 61% were female. Seven-hundred, ninety-five patients (66%) were cared for at one of the first-aid tents. Physicians at the tents assisted in patient management and disposition when crowds restricted ambulance movement. Common complaints included headache (321 patients; 27%), heat-related complaints (148; 12%), nausea or vomiting (91; 7.6%), musculoskeletal complaints (83; 6.9%), and breathing problems (79; 6.6%). Peak activity occurred between 14:00 and 19:00 hours, when 102 patients per hour sought medical attention. Twenty-four patients (0.5/10,000) were transferred to off-site hospitals. CONCLUSIONS: This report on the EMS response, outcomes, and role of the physicians at a large single-day mass gathering may assist EMS planners at future events.

Endotracheal intubation versus supraglottic airway insertion in out-of-hospital cardiac arrest.

OBJECTIVE: To simplify airway management and minimize cardiopulmonary resuscitation (CPR) chest compression interruptions, some emergency medical services (EMS) practitioners utilize supraglottic airway (SGA) devices instead of endotracheal intubation (ETI) as the primary airway adjunct in out-of-hospital cardiac arrest (OHCA). We compared the outcomes of patients receiving ETI with those receiving SGA following OHCA. METHODS: We performed a secondary analysis of data from the multicenter Resuscitation Outcomes Consortium (ROC) PRIMED trial. We studied adult non-traumatic OHCA receiving successful SGA insertion (King Laryngeal Tube, Combitube, and Laryngeal Mask Airway) or successful ETI. The primary outcome was survival to hospital discharge with satisfactory functional status (Modified Rankin Scale ≤3). Secondary outcomes included return of spontaneous circulation (ROSC), 24-h survival, major airway or pulmonary complications (pulmonary edema, internal thoracic or abdominal injuries, acute lung injury, sepsis, and pneumonia). Using multivariable logistic regression, we studied the association between out-of-hospital airway management method (ETI vs. SGA) and OHCA outcomes, adjusting for confounders. RESULTS: Of 10,455 adult OHCA, 8487 (81.2%) received ETI and 1968 (18.8%) received SGA. Survival to hospital discharge with satisfactory functional status was: ETI 4.7%, SGA 3.9%. Compared with successful SGA, successful ETI was associated with increased survival to hospital discharge (adjusted OR 1.40; 95% CI: 1.04, 1.89), ROSC (adjusted OR 1.78; 95% CI: 1.54, 2.04) and 24-h survival (adjusted OR 1.74; 95% CI: 1.49, 2.04). ETI was not associated with secondary airway or pulmonary complications (adjusted OR 0.84; 95% CI: 0.61, 1.16). CONCLUSIONS: In this secondary analysis of data from the multicenter ROC PRIMED trial, ETI was associated with improved outcomes over SGA insertion after OHCA.

The immunomodulatory properties of mesenchymal stem cells and their use for immunotherapy.

There is growing interest in the use of mesenchymal stem cells (MSC) for immune therapy. Clinical trials that use MSC for treatment of therapy resistant graft versus host disease, Crohn's disease and organ transplantation have initiated. Nevertheless, the immunomodulatory effects of MSC are only partly understood. Clinical trials that are supported by basic research will lead to better understanding of the potential of MSC for immunomodulatory applications and to optimization of such therapies. In this manuscript we review some recent literature on the mechanisms of immunomodulation by MSC in vitro and animal models, present new data on the secretion of pro-inflammatory and anti-inflammatory cytokines, chemokines and prostaglandins by MSC under resting and inflammatory conditions and discuss the hopes and expectations of MSC-based immune therapy.

Induction of EAE by T cells specific for alpha B-crystallin depends on prior viral infection in the CNS.

While myelin-reactive T cells are widely believed to play a pathogenic role in multiple sclerosis (MS), no substantial differences appear to exist in T-cell responses to myelin antigens between MS patients and healthy subjects. As an example, indistinguishable peripheral T-cell responses and serum antibody levels have been found in MS patients and healthy controls to alpha B-crystallin, a dominant antigen in MS-affected brain myelin. This suggests that additional factors are relevant in allowing myelin-reactive T cells to become pathogenic. In this study, we examined whether the inflammatory state of the CNS is relevant to the pathogenicity of alpha B-crystallin-specific T cells in mice. In normal mice, T-cell responses against alpha B-crystallin are limited by robust immunological tolerance. Reactive T cells were therefore generated in alpha B-crystallin-deficient mice, and these T cells were transferred into C57BL/6 recipients. While such a transfer in itself never induced any clinical signs of experimental autoimmune encephalomyelitis (EAE) in healthy recipient mice, acute EAE could be induced in animals that had been infected 7 days before with the avirulent A7(74) strain of Semliki Forest virus (SFV). SFV infection alone did not induce clinical disease, nor did it alter the expression levels of the target antigen. Our findings indicate that at least in mice, alpha B-crystallin-specific T cells can trigger EAE but only when prior viral infection has induced an inflammatory state in the CNS that helps recruit and activate T cells.

Tolerization of an established alphaB-crystallin-reactive T-cell response by intravenous antigen.

Tolerance induction to prevent activation of a naïve T-cell repertoire has been well documented in rodents and can be readily achieved by intravenous, oral or intranasal administration of antigen in the absence of adjuvants. In autoimmune diseases such as multiple sclerosis (MS) the presence of an established memory/effector T-cell repertoire against self-antigens is likely to be more relevant than the potential reactivity of naive T cells. Methods to eliminate such an established T-cell response are less well understood. In this study, we explored the effectiveness of intravenous soluble antigen to eliminate a pre-existing T-cell response against alphaB-crystallin, a candidate autoantigen in MS. We used mice that are deficient for the target antigen. This condition allowed for a vigourous T-cell and antibody response to develop upon immunization, and eliminated all possible endogenous mechanisms of tolerance for alphaB-crystallin that are found in normal rodents. When applied 3 weeks after priming with alphaB-crystallin, intravenous administration of soluble antigen almost completely abrogated the established T-cell response in a dose-dependent manner as evidenced by T-cell non-responsiveness in tolerized animals to a re-challenge with antigen in complete Freund's adjuvant. Evaluating delayed-type hypersensitivity responses after tolerance induction revealed that the tolerizing effect was achieved within 24 hr. Furthermore, the tolerizing effect was found to be antigen-specific and long lasting. In contrast, serum antibody levels were markedly increased. Our data clarify that in the absence of any natural form of immune regulation, antigen-specific memory/effector T cells can be effectively silenced by intravenous antigen.