Development and validation of a monocyte activation test for the control/safety testing of an OMV-based meningococcal B vaccine.

It is imperative to ensure biological products are free of contaminating pyrogenic material prior to administration to patients. Historically the rabbit pyrogen test (RPT) was used to screen for such contamination in medicines for intravenous delivery. This test was adapted for use to screen vaccines. However, some, including meningococcal vaccines containing outer membrane vesicles, are intrinsically pyrogenic. Indeed, this is the case for Bexsero which contains relatively high levels of endotoxin and other potential pyrogens such as lipoproteins and porins. The RPT proved a difficult method for measuring the pyrogenic content of Bexsero and differences between laboratories in different countries made repeat testing at the control laboratories problematic resulting in batches being wrongly identified as unsafe. At NIBSC a monocyte activation test (MAT) was adapted and validated as an alternative. This required setting of a specification in-house and deciding on a decisional procedure using multiple donors, allowing batches equally pyrogenic or less, than those batches shown to be safe in a clinical trial, to be certified as safe. The resulting format was a reference comparison method with an upper limit of 1.8 relative pyrogen units (RPU). The batch passed if an initial four donors had a response equal to or less than 1.8 RPU, if one donor is above this limit the batch was tested in a further four donors and seven of the eight must be equal to or below 1.8 RPU. If two donors have a response greater than 1.8 the batch failed.

Evaluation of the monocyte activation test for the safety testing of meningococcal B vaccine Bexsero: A collaborative study.

The aim of this collaborative study was to evaluate the robustness of the monocyte activation test (MAT) for quantifying the pyrogenic content in the outer membrane vesicle (OMV)-containing vaccine Bexsero: the first meningococcal B vaccine to be licenced. We analysed datasets from 9 laboratories covering 15 test systems for 3 batches of Bexsero with higher, equivalent and lower activity relative to a reference lot in the MAT. Activity was measured in terms of relative pyrogen units (RPU) based on European Pharmacopoeia (Ph. Eur.) MAT Chapter 2.6.30 Method C: Reference Lot Comparison Test. We report that all 15 test systems were consistent in that they showed sample A to be the most active in the MAT; that 13 of 15 test systems had an accuracy of more than 80% and an overall geometric mean RPU of 1.03 with lower and upper 95% confidence limits of 0.97 and 1.09 respectively for a sample with an expected value of 1.00 RPU. We also report larger variability in the results for test systems involving cells from individual blood donations for sample A suggesting that there could be donor to donor differences in sensitivity to the vaccine constituents responsible for the higher activity of this batch. Overall, the consistency and accuracy of the MAT was remarkable given the range of test systems used by participants, all of which are permitted by the Ph. Eur. General MAT Chapter. This is important given the limitations of the rabbit pyrogen test for the control of pyrogenicity in general and particularly with products with intrinsic pyrogenicity such as Bexsero.

Monocyte-activation test to reliably measure the pyrogenic content of a vaccine: An in vitro pyrogen test to overcome in vivo limitations.

Pyrogen content is one of the critical quality attributes impacting the safety of a product, and there is an increasing need for assays that can reliably measure this attribute in vaccines. The Limulus amebocyte lysate (LAL) assay and the rabbit pyrogen test (RPT) are the canonical animal-based pyrogen tests currently used to release vaccines; however, there are several drawbacks associated with these tests when applied to Bexsero, intrinsically pyrogenic product, containing a meningococcal Outer Membrane Vesicle component. While the RPT, as applied to Bexsero at its given dilution, ensures safe vaccine, it is highly variable and prone to false positive results. On the other hand, the LAL assay although quantitative, can detect only endotoxin pyrogens and is not sufficient for monitoring the safety of Bexsero, which contains both LPS and non-endotoxin pyrogens. Being aware of these limitations of the RPT and LAL when applied to Bexsero, the Monocyte Activation Test (MAT) which is sensitive to both endotoxin and non-endotoxin based pyrogens has been developed as an alternative pyrogen test. Here, the development and the validation of a MAT assay adapted from the European pharmacopoeia for Bexsero, is described. The MAT assay is then used for monitoring the safety and consistency of Bexsero vaccines at release, providing great advantages in terms of reduced variability with respect to RPT, reduction of animal use, in line with the 3Rs principle concerning the protection of animals and faster time to market. In addition the correlation of the MAT to the RPT has been demonstrated supporting the replacement of the in vivo method and the potential application of the assay to other intrinsically pyrogenic vaccines.

Material-mediated pyrogens in medical devices: Applicability of the in vitro Monocyte Activation Test.

Pyrogenicity presents a challenge to clinicians, medical device manufacturers, and regulators. A febrile response may be caused by endotoxin contamination, microbial components other than endotoxin, or chemical agents that generate a material-mediated pyrogenic response. While test methods for the assessment of endotoxin contamination and some microbial components other than endotoxin are well-established, material-mediated pyrogens remain elusively undefined. This review presents the findings of literature searches conducted to identify material-mediated pyrogens associated with medical devices. The in vivo rabbit pyrogen test (RPT) is considered to be the "gold standard" for medical device pyrogenicity testing, despite the fact that few medical device-derived material-mediated pyrogens are known. In line with global efforts to reduce the use of research animals, an in vitro monocyte activation test (MAT) has the potential to replace the RPT. The MAT is used to detect substances that activate human monocytes to release cytokines. This review will also describe the potential opportunities and challenges associated with MAT adoption for the detection of material-mediated pyrogens in medical device testing.

Assessment of pyrogenic response of lipoteichoic acid by the monocyte activation test and the rabbit pyrogen test.

Lipoteichoic acid (LTA) is a non-endotoxin pyrogen of a great importance in the pathogenesis of sepsis. The Rabbit Pyrogen Test (RPT) is able to detect all types of pyrogens but involves the use of animals. The Bacterial Endotoxin Test (BET) cannot fully replace the RPT because it only detects endotoxins. The Monocyte Activation Test (MAT) is sensitive to all types of pyrogens and it is based on the same biological mechanism that is responsible for the fever reaction in humans. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has recommended its use for other pyrogens than endotoxin because its equivalence to RPT can be demonstrated. The aim of this study was to evaluate the pyrogenic responses of the RPT and MAT that was induced by LTA. Different LTA concentrations were assayed by the MAT in parallel to the RPT. The results showed that the MAT was more sensitive than the RPT, demonstrating that the MAT detected LTA. This result may contribute to the acceptance of this test by the Brazilian regulatory agencies as a replacement for the animals used in the RPT.

Investigation of interleukin-1ß release from cryopreserved blood stimulated with endotoxin.

The feasibility of an indigenously developed ELISA method to determine cytokine response to wide spectrum of pyrogenic stimuli utilizing fresh human whole blood is limited by the availability of healthy donors. The possibility of using cryopreservation of pooled human blood for detection of cytokine response to lipopolysaccharide is explored in this study. The effect of cryopreservation on blood parameters, cellular morphology and cytokine response were compared with that of the pooled fresh blood and cryopreserved blood from single and multiple donors. In vitro pyrogenic stimulation with 0.5 and 5 EU of LPS was monitored on fresh and cryopreserved pooled blood from single and multiple donors. The release of IL-1ß was quantitated by Sandwich ELISA (1, 10, 25, 45 and 75 days) after storage. The results indicated that the cryopreserved blood displayed enhanced IL-1ß release on stimulation with LPS, when compared to fresh blood. The maximum release of IL-1ß level was observed at 2h when 5 EU of LPS was treated with pooled fresh blood which is similar to that of fresh blood. After 75 days storage of pooled cryopreserved blood the IL-1ß release was maximum at 9 and 15 h when treated with 5 and 0.5 EU of LPS. Observations of the study suggest that cryopreserved pooled blood is an economically and experimentally viable alternative to fresh blood. This investigative study promises short term storage and regular supply of non-allergic, pathogen free human blood for the detection of interleukin-1ß for the evaluation of in vitro pyrogenicity.

Indomethacin abolishes core temperature, but not cardiovascular or renal, responses to lipopolysaccharide in conscious lambs.

1. Core temperature (Tc), cardiovascular and renal responses to lipopolysaccharide (LPS), as well as the role of endogenously produced prostaglandins (PG) in influencing these responses, were investigated in the present study in conscious, chronically instrumented lambs. 2. Core temperature, mean arterial pressure, heart rate (HR), renal blood flow (RBF) and several parameters of renal function were measured for 30 min before and for 5 h after intravenous injection of 0.03 µg/kg of the LPS Salmonella abortus equi (n = 9) or saline vehicle (n = 9). 3. After injection of LPS, Tc increased with a latency of 40 min, duration of 130 min and magnitude of 1.5°C. Mean arterial pressure increased within 110 min of LPS injection and then decreased below baseline within 5 h, concomitant with an increase in HR. There was a sustained increase in RBF after LPS injection and a significant increase in urinary flow rate, as well as Na(+) and Cl(-) excretion. 4. To determine the role of PGs in the responses to LPS observed, additional experiments were performed in another group of conscious lambs that had been pretreated with the non-selective cyclo-oxygenase inhibitor indomethacin (10 mg/kg; n = 6). 5. Although indomethacin abolished the Tc response to LPS, it had no significant effect on the cardiovascular and renal responses to LPS. There were no effects of saline vehicle on any of the variables measured. 6. These data provide evidence that, in conscious young lambs, cardiovascular and renal responses to LPS do not appear to be mediated by endogenously produced PGs and that they are independent of pyrogen-induced changes in Tc.

Acute-phase responses vary with pathogen identity in house sparrows (Passer domesticus).

Pathogens may induce different immune responses in hosts contingent on pathogen characteristics, host characteristics, or interactions between the two. We investigated whether the broadly effective acute-phase response (APR), a whole body immune response that occurs in response to constitutive immune receptor activation and includes fever, secretion of immune peptides, and sickness behaviors such as anorexia and lethargy, varies with pathogen identity in the house sparrow (Passer domesticus). Birds were challenged with a subcutaneous injection of either a glucan at 0.7 mg/kg (to simulate fungal infection), a synthetic double-stranded RNA at 25 mg/kg (to simulate viral infection), or LPS at 1 mg/kg (to simulate a gram-negative bacterial infection), and then body mass, core body temperature changes, sickness behaviors, and secretion of an acute-phase protein, haptoglobin, were compared. Despite using what are moderate-to-high pyrogen doses for other vertebrates, only house sparrows challenged with LPS showed measurable APRs. Febrile, behavioral, and physiological responses to fungal and viral mimetics had minimal effects.

Comparison of temperature rise interpretations in the rabbit pyrogen test among Chinese, Japanese, European, and United States pharmacopeias and 2-2-2 theoretical models proposed by S. Hoffmann.

Although the rabbit pyrogen test is one of the crucial methods included in each pharmacopeia to evaluate the safety of parenteral medicine, the experimental procedures and pyrogen result judgment algorithms (PRJAs) are still greatly different from one another. In the first stage of testing, original data of 879 batches from a total of 2637 rabbits in our laboratory were judged by PRJAs in the Chinese Pharmacopoeia 2005 III, the Japanese Pharmacopoeia XIV, the Japanese Pharmacopoeia XV, the European Pharmacopeia 6.0, the United States Pharmacopoeia 32 NF27 and two theoretical models proposed by S. Hoffmann, respectively. The results were analyzed to evaluate the effects of various PRJAs. It was shown that: (i) the significant differences in the results judged by various pharmacopeias and Hoffmann's theoretical models were mainly due to the PRJAs and the great differences in PRJAs should be harmonized throughout the world based on balance of reducing animal use and guaranteeing the safety of medicines; (ii) it is better to use PRJAs that depend on the threshold of the sum of temperature rise of all tested rabbits than those that depend on the number of rabbits that are over the threshold of temperature rise of individual rabbit according to clinical proof and the experimental data; and (iii) the PRJA of the Japanese Pharmacopoeia XV has obvious advantages when the total suspicious rate of samples was less than 10%. Additionally, a new PRJA designed for reducing the additional experiment stages and animal consumption is promoted for evaluation.

Busca ativa de possíveis causas de pirogenia em pacientes submetidos a procedimentos coronários diagnósticos e terapêuticos / Active search of pyrogen reaction sources in patients undergoing diagnostic and therapeutic coronary procedures

Introdução: A reação pirogênica é desencadeada pela presença, na corrente sanguínea, de soluções contaminadas, infundidas no paciente, contendo endotoxinas ou produtos de degradação proteica. Apesar da baixa incidência, seu foco deve ser investigado e erradicado, pois essas ocorrências podem ser deletérias e refletem a qualidade do serviço prestado, já que a causa pode ser proveniente de fatores relacionados ao exame, às soluções utilizadas ou ao processo de esterilização. Método: Estudo prospectivo avaliando pacientes submetidos a procedimentos coronários percutâneos diagnósticos ou terapêuticos que desenvolveram reação pirogênica. Essa amostra foi comparada a um grupo controle de pacientes consecutivos que não apresentaram a referida intercorrência, sendo a possível causa pesquisada por meio do uso de um protocolo tipo check list...

Background: Pyrogen reactions are triggered by intravenous injection of fluids or medications contaminated by bacterial endotoxins. Despite its low incidence, it must be investigated and eliminated, as it may be harmful to the patient and reflects the quality of the catheterization laboratory. Methods: Prospective study to evaluate patients undergoing diagnostic or therapeutic percutaneous coronary procedures who develop pyrogen reactions. This group was compared with a historical cohort of patients undergoing invasive procedures with no pyrogen reactions. The possible causes of pyrogen reactions were established based on the use of a check-list protocol...

Physiology of fever.

Knowing how the body reacts to the presence of pathogens allows healthcare professionals to make informed decisions about what action to take in caring for the child with fever. A raised body temperature raises the metabolic rate and makes the immune response more efficient. It also stimulates naturally occurring anti-pyretics but can also have harmful effects. Careful monitoring based on risk of serious illness is recommended in new guidelines on the management of feverish illness in young children provide (National Institute of Health and Clinical Excellence (NICE 2007), which also provide an opportunity for standardising fever management.

Fever and circulating cytokines induced by double-stranded RNA in guinea pigs: dependence on the route of administration and effects of repeated injections.

AIMS: The aim of this study was to characterize the properties of synthetic double-stranded RNA to induce fever and circulating cytokines in guinea pigs with special emphasis on the route of administration and on a putative development of tolerance to this pyrogen. METHODS: Changes in abdominal temperature were recorded in unrestrained animals by use of intra-abdominally implanted radiotransmitters. Circulating concentrations of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were measured by use of specific bioassays. RESULTS: The pyrogenic effect of double-stranded RNA at a dose of 500 microg kg(-1) depended on the route of its administration. Intra-arterial (i.a.) or intraperitoneal injections of double-stranded RNA induced pronounced fevers and strong elevations of circulating TNF-alpha and IL-6. Intramuscular injections of the synthetic pyrogen caused rather moderate febrile and cytokine responses. Administration of synthetic RNA into artificial subcutaneously implanted Teflon chambers had no pyrogenic and cytokine-inducing effects. I.a. injections of double-stranded RNA, repeated five times at intervals of 3 days, resulted in fevers of similar shape and duration and similar cytokine response patterns. However, the strength of fever and cytokine formation was significantly reduced, although not abolished, in response to the repeated injections compared with the first injection, indicating a partial development of tolerance. CONCLUSIONS: The modulation of the strength of RNA-induced fever, dependent on the route of administration, or the state of partial tolerance to this pyrogen, may thus be related to the formation of pyrogenic cytokines.

Differential sensitivities of pyrogenic chemokine fevers to CC chemokine receptor 5 antibodies.

Macrophage inflammatory protein (MIP)-1beta and RANTES (regulated on activation, normal T-cells expressed and secreted) are members of the CC-family of chemokines. Although these two peptides are structurally and functionally related to one another, each exhibits distinct features, which allows it to independently regulate specific aspects of the host inflammatory response. They evoked intense and functionally different febrile responses when applied directly on pyrogen-sensitive cells located in the in the preoptic area of the anterior hypothalamus (POA). The present experiments were carried out to test the central role of CCR5, a functional receptor for MIP-1beta and RANTES, in the febrile responses induced by these chemokines when injected directly into the POA. The microinjection of an equimolecular dose (50 pg) of either MIP-1beta or RANTES into the POA induced a rapid onset; monophasic fever in rats that persisted for a long period. The microinjection of 2.0 microg specific neutralizing antibodies against CCR5 (anti-CCR5) into the POA fails to affect the effects on body temperature induced by MIP-1beta. However, pretreatment with the same dose of anti-CCR5 suppressed the febrile response induced by RANTES given at the same site. The microinjection of control IgG or anti-CCR5 does not affect basal temperature, when administered alone at the same hypothalamic site. The present experiments show that hypothalamic CCR5 are functionally involved in the febrile response induced by RANTES, but not by MIP-1beta. They also suggest the existence of functionally different components in the presumptive primary locus of the thermoregulatory controller, in which both chemotactic cytokines, together other mediators, could play a relevant role in the complex process of fever pathogenesis.

Endotoxins and medical devices: the significance of dead bacteria.

There has been some recent speculation that endotoxins, the toxic molecules that are present in the cell wall of certain bacteria, could play a role in the development of tissue reactions to medical devices. This article discusses the basis for this speculation.

Association of interleukin-6 in the cerebrospinal fluid during crisis of calf with ammoniated feed syndrome.

Ammoniated feed syndrome (AFS) in cattle is a neurotoxic syndrome caused by feeding specific ammoniated forage. To clarify the pathophysiology of AFS, we examined the association of interleukin-6 (IL-6) in the brain. By feeding milk either from cows fed such ammoniated forage or milk added with 4-methyl-imidazole, newborn calves showed a neurotoxic crisis of hyperexcitability, ataxia, muscle tremor, circling, roaring, epileptoid seizure, sweating and marked fever response. Although these calves had no pathological lesions in the brain, we detected a rise in IL-6 in the cerebrospinal fluid (CSF). Tumor necrosis factor-alpha (TNF-alpha) was not detected in the CSF. In the sera, IL-6 and TNF-alpha hardly changed during the experiment. Administration of recombinant human IL-6 into the lateral ventricle resulted in fever. Thus, we believe IL-6 in the CSF is related to the fever response in newborn calves with AFS.

Catheter related bacterial infections mimic reactions to exogenous pyrogens during hemodialysis.

Pyrogenic reactions are characterized by fever, chills, hypotension, or a combination of these developing during or shortly after hemodialysis in a previously asymptomatic patient. The temporal association with treatment implicates exposure of the patient's blood to bacterial pyrogens from contaminated dialysate or a reused dialyzer. Routine body temperature monitoring is recommended to detect these exposures. The current study was prompted by the appearance of several symptomatic febrile episodes in patients who were asymptomatic and afebrile before treatment with high-flux hemodialysis. During a 6 month period, temperatures were measured with a digital oral thermometer before and after 9,605 high-flux hemodialyses in 163 patients. Elevations above 100 degrees F (37.8 degrees C) were observed during or after 33 dialyses in 15 patients. In 18 of these dialyses, the temperature was also elevated before treatment began. Four patients who had no symptoms or fever before dialysis accounted for febrile reactions during 11 of the remaining 15 dialysis treatments. Fever was accompanied by rigors during most of the episodes. Subsequent blood cultures grew Enterococcus faecalis (two), Enterobacter cloacae (two), and Pseudomonas aeruginosa and cepacia (one). All four patients had indwelling silastic double lumen venous catheters (PermCaths), all responded to intravenous antibiotics, and all required eventual removal of the catheter. The apparent precipitation of sepsis by dialysis indicates that shear forces caused by high pulsatile blood flow through the catheter may dislodge organisms that have colonized the lumen. Intraluminal instillation of antibiotics is suggested as a preventative measure.

A new procedure for stable quantification of endotoxin in dialysate fluid using Limulus reagent.

For reliable determination of endotoxin, the activation of enzymes in lysate before measurement should be prevented, and the authors have designed a new procedure to effect this by dissolving the enzymes in lysate in a buffer solution of low pH. A given amount of the enzymes in lysate was dissolved in a lower pH buffer solution (pH 6.1-6.3) and the substrate was dissolved in a higher pH buffer solution (pH 8.0). After standing for 0-24 hr, both solutions were mixed with the sample solution. Data on blank absorbance and calibration line slope obtained by the new procedure were compared with those obtained by the conventional procedure. In the conventional procedure, blank absorbance increased with standing time, reaching approximately seven times the initial value in 24 hr, whereas in the improved procedure, it increased by 1.5 times at a standing time of 3 hr, after which it was independent of standing time. The change in slope of the calibration line with standing time was more gradual in the improved procedure than in the conventional procedure. The authors conclude that the activation of enzymes in lysate can be prevented by dissolving the enzymes in a buffer solution of low pH, and that this procedure is effective for long-term monitoring of endotoxin concentration.

Influence of perflubron emulsion particle size on blood half-life and febrile response in rats.

Perfluorochemical (PFC) emulsions are particulate in nature and, as such, can cause delayed febrile reactions when injected intravenously. This study investigated the influence of emulsion particle size on intravascular retention and on body temperature changes in unrestrained conscious rats. Concentrated (60% to 90% w/v) emulsions based on perflubron (perfluorooctyl bromide [PFOB]) with mean particle sizes ranging from 0.05 microns to 0.63 microns were tested. Rats were fitted with a chronic jugular catheter and an abdominal body temperature telemetry unit. Fully recovered, conscious rats were monitored for 24 hours after infusion (dose = 2.7 g PFC/kg). Emulsion blood half-life (T1/2) was determined from blood perflubron levels measured by gas chromatography. Emulsions with a particle size of 0.2-0.3 microns caused fevers (6 to 8 hour duration) which peaked at 1-1.5 degrees C above normal (approximately 37.5 degrees C). Fevers could be blocked by i.v. treatment with either cyclooxygenase inhibitors (ibuprofen) or corticosteroids (dexamethasone). Both intensity and duration of the temperature response, quantified by area under the temperature curve, was decreased significantly for emulsions with a particle size < or = 0.12 micron. Blood T1/2 varied inversely with particle size, and was 3 to 4 fold longer for emulsions with a mean particle size < or = 0.2 micron. Thus, smaller emulsion particles more effectively evaded the reticuloendothelial system, which resulted in longer intravascular retention, less macrophage activity, and reduced febrile responses.

[Blood plasma diene conjugates during the stimulation and inhibition of lipid peroxidation in dogs with different forms of healing in experimental myocardial infarct]. / Dienovye kon''iugaty plazmy krovi pri stimuliatsii i ingibirovanii perekisnogo okisleniia lipidov u sobak s razlichnymi formami zazhivleniia éksperimental'nogo infarkta miokarda.

In experiments on 45 dogs with a model of normo-, hyper-, and hyporeactive myocardial infarction, (MI) lipid peroxidation (LP) was stimulated by ultraviolet irradiation of autologous blood and inhibited by alpha-tocopherol acetate. It is shown that changes of LP are characteristic of uncomplicated healing of normoreactive MI. Hyperactivation of LP in hyperreactive MI and its hypoactivation in hyporeactive MI are among the factors of complication of its healing by postinfarction aneurysm of the heart. LP modulation consisting in bringing its changes in conformity with those in uncomplicated forms of the disease facilitates normal MI healing with the formation of a postinfarction scar. Antioxidants are indicated in hyperreactive MI, and LP activation by means of stimulators is recommended in hyporeactive MI.