Riding the waves from epidemic to endemic: Viral mutations, immunological change and policy responses.

Nonpharmaceutical interventions (NPI) are an important tool for countering pandemics such as COVID-19. Some are cheap; others disrupt economic, educational, and social activity. The latter force governments to balance the health benefits of reduced infection and death against broader lockdown-induced societal costs. A literature has developed modeling how to optimally adjust lockdown intensity as an epidemic evolves. This paper extends that literature by augmenting the classic SIR model with additional states and flows capturing decay over time in vaccine-conferred immunity, the possibility that mutations create variants that erode immunity, and that protection against infection erodes faster than protecting against severe illness. As in past models, we find that small changes in parameter values can tip the optimal response between very different solutions, but the extensions considered here create new types of solutions. In some instances, it can be optimal to incur perpetual epidemic waves even if the uncontrolled infection prevalence would settle down to a stable intermediate level.

The hammer and the jab: Are COVID-19 lockdowns and vaccinations complements or substitutes?

The COVID-19 pandemic has devastated lives and economies around the world. Initially a primary response was locking down parts of the economy to reduce social interactions and, hence, the virus' spread. After vaccines have been developed and produced in sufficient quantity, they can largely replace broad lock downs. This paper explores how lockdown policies should be varied during the year or so gap between when a vaccine is approved and when all who wish have been vaccinated. Are vaccines and lockdowns substitutes during that crucial time, in the sense that lockdowns should be reduced as vaccination rates rise? Or might they be complementary with the prospect of imminent vaccination increasing the value of stricter lockdowns, since hospitalization and death averted then may be permanently prevented, not just delayed? We investigate this question with a simple dynamic optimization model that captures both epidemiological and economic considerations. In this model, increasing the rate of vaccine deployment may increase or reduce the optimal total lockdown intensity and duration, depending on the values of other model parameters. That vaccines and lockdowns can act as either substitutes or complements even in a relatively simple model casts doubt on whether in more complicated models or the real world one should expect them to always be just one or the other. Within our model, for parameter values reflecting conditions in developed countries, the typical finding is to ease lockdown intensity gradually after substantial shares of the population have been vaccinated, but other strategies can be optimal for other parameter values. Reserving vaccines for those who have not yet been infected barely outperforms simpler strategies that ignore prior infection status. For certain parameter combinations, there are instances in which two quite different policies can perform equally well, and sometimes very small increases in vaccine capacity can tip the optimal solution to one that involves much longer and more intense lockdowns.

Cervical arthroplasty in a professional kick-boxing fighter, 7 years follow-up.

Spinal surgery in professional athletes is a topic of much discussion. Anterior cervical discectomy and fusion (ACDF) is the standard procedure used by surgeons, and other techniques used to treat athletes includes foraminotomies, laminoplasties and total disc replacement. Total disc replacement is an unusual technique used to treat athletes in general and is becoming a more important issue in full contact sports. This case report illustrates a 34 years old professional fighter that suffered a cervical injury that evolved with cervical axial pain and irradiated pain and numbness. She was submitted to total disc replacement (TDR) at the C5-6 level, returning to competitive sports after and with a seven-year follow-up. To the date she remains symptom free and besides having an anterior foramen, the spine was able to keep movement at that level. TDR may be a safe and trustworthy technique when treating elite athletes.

La cirugía de columna en atletas profesionales es un tema de mucha discusión. La discectomía y fusión cervical anterior es el procedimiento estándar utilizado por los cirujanos, y otras técnicas utilizadas para tratar a los atletas incluyen foraminotomías, laminoplastías y reemplazo total de disco. El reemplazo total del disco es una técnica inusual utilizada para tratar a los atletas en general y se está convirtiendo en un tema más importante en los deportes de contacto completo. Este informe de caso ilustra a una luchadora profesional de 34 años que sufrió una lesión cervical que evolucionó con dolor axial cervical y dolor irradiado y entumecimiento. Fue sometida a colocación de prótesis de disco en el nivel C5-6, regresando a los deportes competitivos y con un seguimiento de siete años. Hasta la fecha permanece libre de síntomas y además de tener un foramen anterior, la columna vertebral fue capaz de mantener el movimiento a ese nivel. La cirugía puede ser una técnica segura y confiable cuando se trata a atletas de élite.

Cervical arthroplasty in a professional kick-boxing fighter, 7 years follow-up / Artroplastia cervical en un luchador profesional de kick-boxing, 7 años de seguimiento

Abstract: Spinal surgery in professional athletes is a topic of much discussion. Anterior cervical discectomy and fusion (ACDF) is the standard procedure used by surgeons, and other techniques used to treat athletes includes foraminotomies, laminoplasties and total disc replacement. Total disc replacement is an unusual technique used to treat athletes in general and is becoming a more important issue in full contact sports. This case report illustrates a 34 years old professional fighter that suffered a cervical injury that evolved with cervical axial pain and irradiated pain and numbness. She was submitted to total disc replacement (TDR) at the C5-6 level, returning to competitive sports after and with a seven-year follow-up. To the date she remains symptom free and besides having an anterior foramen, the spine was able to keep movement at that level. TDR may be a safe and trustworthy technique when treating elite athletes.

Resumen: La cirugía de columna en atletas profesionales es un tema de mucha discusión. La discectomía y fusión cervical anterior es el procedimiento estándar utilizado por los cirujanos, y otras técnicas utilizadas para tratar a los atletas incluyen foraminotomías, laminoplastías y reemplazo total de disco. El reemplazo total del disco es una técnica inusual utilizada para tratar a los atletas en general y se está convirtiendo en un tema más importante en los deportes de contacto completo. Este informe de caso ilustra a una luchadora profesional de 34 años que sufrió una lesión cervical que evolucionó con dolor axial cervical y dolor irradiado y entumecimiento. Fue sometida a colocación de prótesis de disco en el nivel C5-6, regresando a los deportes competitivos y con un seguimiento de siete años. Hasta la fecha permanece libre de síntomas y además de tener un foramen anterior, la columna vertebral fue capaz de mantener el movimiento a ese nivel. La cirugía puede ser una técnica segura y confiable cuando se trata a atletas de élite.

Navigated Transtubular Extraforaminal Decompression of the L5 Nerve Root at the Lumbosacral Junction: Clinical Data, Radiographic Features, and Outcome Analysis.

Purpose. Extraforaminal decompression of the L5 nerve root remains a challenge due to anatomic constraints, severe level-degeneration, and variable anatomy. The purpose of this study is to introduce the use of navigation for transmuscular transtubular decompression at the L5/S1 level and report on radiological features and clinical outcome. Methods. Ten patients who underwent a navigation-assisted extraforaminal decompression of the L5 nerve root were retrospectively analyzed. Results. Six patients had an extraforaminal herniated disc and four had a foraminal stenosis. The distance between the L5 transverse process and the para-articular notch of the sacrum was 12.1 mm in patients with a herniated disc and 8.1 mm in those with a foraminal stenosis. One patient had an early recurrence and another developed dysesthesia that resolved after 3 months. There was a significant improvement from preoperative to postoperative NRS with the results being sustainable at follow-up. ODI was also significantly improved after surgery. According to the Macnab grading scale, excellent or good outcomes were obtained in 8 patients and fair ones in 2. Conclusions. The navigated transmuscular transtubular approach to the lumbosacral junction allows for optimal placement of the retractor and excellent orientation particularly for foraminal stenosis or in cases of complex anatomy.

First report of mecC MRSA in human samples from Austria: molecular characteristics and clinical data.

Reports of mecC methicillin-resistant Staphylococcus aureus (MRSA) strains have been published from several European countries. We describe the first six mecC MRSA isolates of human origin from Austria and report the application of a rapid PCR test. Candidate isolates (n = 295) received between 2009 and 2013 were investigated phenotypically by cefoxitin screening and streaking on ChromID MRSA plates. The presence of mecC was confirmed in six isolates from blood cultures, wound swabs and screening samples of four female and two male patients (age range 7-89 years) by an in-house PCR method and the new Genspeed MRSA test (Greiner Bio-One, Kremsmünster, Austria). The mecC MRSA were further characterized by whole genome sequencing, multilocus sequence and spa typing. Antimicrobial susceptibility testing was performed by Eucast disk-diffusion method and Vitek 2. The six mecC MRSA isolates were from two clonal lineages (CC130, including a new single-locus variant, and CC599) and four different spa types (t843, t1535, t3256, t5930). Analysis for virulence factor genes yielded lukED, eta, etd2 and edin-B (CC130 isolates) and tst, lukED, eta and sel (ST599 isolates). The Genspeed MRSA test identified mecC in all isolates whereas Vitek 2 failed to detect methicillin resistance in one isolate. The strains were susceptible to a wide range of non-ß-lactam antibiotics. All patients were successfully treated or decolonized. mecC MRSA are present in Austria as colonizers but may also cause infections. Thus, laboratories must choose appropriate test methods such as cefoxitin screening and confirmation using molecular assays specifically targeting mecC.

Cell therapy for intervertebral disc repair: advancing cell therapy from bench to clinics.

Intervertebral disc (IVD) degeneration is a major cause of pain and disability; yet therapeutic options are limited and treatment often remains unsatisfactory. In recent years, research activities have intensified in tissue engineering and regenerative medicine, and pre-clinical studies have demonstrated encouraging results. Nonetheless, the translation of new biological therapies into clinical practice faces substantial barriers. During the symposium "Where Science meets Clinics", sponsored by the AO Foundation and held in Davos, Switzerland, from September 5-7, 2013, hurdles for translation were outlined, and ways to overcome them were discussed. With respect to cell therapy for IVD repair, it is obvious that regenerative treatment is indicated at early stages of disc degeneration, before structural changes have occurred. It is envisaged that in the near future, screening techniques and non-invasive imaging methods will be available to detect early degenerative changes. The promises of cell therapy include a sustained effect on matrix synthesis, inflammation control, and prevention of angio- and neuro-genesis. Discogenic pain, originating from "black discs" or annular injury, prevention of adjacent segment disease, and prevention of post-discectomy syndrome were identified as prospective indications for cell therapy. Before such therapy can safely and effectively be introduced into clinics, the identification of the patient population and proper standardisation of diagnostic parameters and outcome measurements are indispensable. Furthermore, open questions regarding the optimal cell type and delivery method need to be resolved in order to overcome the safety concerns implied with certain procedures. Finally, appropriate large animal models and well-designed clinical studies will be required, particularly addressing safety aspects.

Integrating stochastic time-dependent travel speed in solution methods for the dynamic dial-a-ride problem.

In urban areas, logistic transportation operations often run into problems because travel speeds change, depending on the current traffic situation. If not accounted for, time-dependent and stochastic travel speeds frequently lead to missed time windows and thus poorer service. Especially in the case of passenger transportation, it often leads to excessive passenger ride times as well. Therefore, time-dependent and stochastic influences on travel speeds are relevant for finding feasible and reliable solutions. This study considers the effect of exploiting statistical information available about historical accidents, using stochastic solution approaches for the dynamic dial-a-ride problem (dynamic DARP). The authors propose two pairs of metaheuristic solution approaches, each consisting of a deterministic method (average time-dependent travel speeds for planning) and its corresponding stochastic version (exploiting stochastic information while planning). The results, using test instances with up to 762 requests based on a real-world road network, show that in certain conditions, exploiting stochastic information about travel speeds leads to significant improvements over deterministic approaches.

Temocillin and meropenem to discriminate resistance mechanisms leading to decreased carbapenem susceptibility with focus on OXA-48 in Enterobacteriaceae.

A temocillin minimal inhibitory concentration ≥ 128 mg/L combined with the results of meropenem double disc synergy testing was used to (i) discriminate carbapenemase production from other resistance mechanisms leading to decreased carbapenem susceptibility; and (ii) differentiate Ambler classes in carbapenemase-producing enterobacteriaceae (CPE). The suggested test algorithm discriminated all extended spectrum ß-lactamase/AmpC from CPE isolates, which could further be divided correctly into Ambler classes A and B enzymes as well as OXA-48 in all cases. The algorithm is simple to implement as part of the daily routine in a standard microbiology laboratory with limited access to or resources for molecular biological tools.

Bone and cellular immune system of multiparous sows are insensitive to ovariectomy and nutritive calcium shortage.

Research in osteoporosis, which is a complex systemic disease, demands suitable large animal models. In pigs, most research has been done in growing minipigs, which probably are not ideal models for postmenopausal osteoporosis. Therefore, our aim was to analyze the effects of ovariectomy (OVX) and nutritive calcium shortage on multiparous Large White sows. 32 animals were randomly assigned to 4 groups in a cross design with OVX vs. sham and physiological calcium supplementation (0.75% calcium) vs. dietary calcium shortage (0.3% calcium). The observation period was 10 months with blood sampling every 2 months for hematological, immunological, and biochemical bone marker measurements. At the termination of the experiment, animals were sacrificed. Samples of trabecular bone of distal radius, proximal tibia, and sixth lumbar vertebra were subjected to micro-computed tomography imaging and ashed afterwards. Dual X-ray absorptiometry scans of the proximal femora were performed with prepared bones being placed in a water bath for mimicking soft tissue. Analyses of bone marker and cytokine profile kinetics, distribution of leukocyte subpopulations, and morphometrical and densitometrical analyses showed no evidence of any impact of OVX or calcium shortage. In conclusion, the skeleton of adult sows of a conventional breed is seemingly protected from effects of OVX and calcium shortage.

Metaheuristics for the dynamic stochastic dial-a-ride problem with expected return transports.

The problem of transporting patients or elderly people has been widely studied in literature and is usually modeled as a dial-a-ride problem (DARP). In this paper we analyze the corresponding problem arising in the daily operation of the Austrian Red Cross. This nongovernmental organization is the largest organization performing patient transportation in Austria. The aim is to design vehicle routes to serve partially dynamic transportation requests using a fixed vehicle fleet. Each request requires transportation from a patient's home location to a hospital (outbound request) or back home from the hospital (inbound request). Some of these requests are known in advance. Some requests are dynamic in the sense that they appear during the day without any prior information. Finally, some inbound requests are stochastic. More precisely, with a certain probability each outbound request causes a corresponding inbound request on the same day. Some stochastic information about these return transports is available from historical data. The purpose of this study is to investigate, whether using this information in designing the routes has a significant positive effect on the solution quality. The problem is modeled as a dynamic stochastic dial-a-ride problem with expected return transports. We propose four different modifications of metaheuristic solution approaches for this problem. In detail, we test dynamic versions of variable neighborhood search (VNS) and stochastic VNS (S-VNS) as well as modified versions of the multiple plan approach (MPA) and the multiple scenario approach (MSA). Tests are performed using 12 sets of test instances based on a real road network. Various demand scenarios are generated based on the available real data. Results show that using the stochastic information on return transports leads to average improvements of around 15%. Moreover, improvements of up to 41% can be achieved for some test instances.

Iso-C/3-dimensional neuronavigation versus conventional fluoroscopy for minimally invasive pedicle screw placement in lumbar fusion.

BACKGROUND: Minimally invasive spinal surgery (MISS) has evolved over the past years due to the combination of microsurgery, minimal access strategies and neuronavigation. Percutaneous or mini-open pedicle screw placement is a challenge, especially for surgeons not experienced in the use of biplanar fluoroscopy or stereotactic imaging techniques. The aim of the study was to compare the precision of pedicle screw fixation for lumbar fusion procedures using Iso-C/stereotactic 3D neuronavigation (3D NAV) vs. standard AP/lateral fluoroscopy (2D NAV). METHODS: Our first 42 patients undergoing MISS for one- or 2-level lumbar or lumbosacral fusion procedures for degenerative lumbar pathology were included in this study. Either 3D NAV (n=29) or standard fluoroscopy (n=13) was used to aid screw placement. Demographics, operative time, blood loss, and screw placement accuracy were evaluated. Screw placement was evaluated postoperatively using lumbar CT scanning. Accuracy of 3D NAV was evaluated by comparing intraoperative planning screenshots to postoperative CT placement of screws. RESULTS: There were no significant differences between groups for mean age, gender or intraoperative blood loss. 90.9% of 3D NAV screws and 73.7% of fluoroscopy screws had no pedicle perforation (p=0.04). Intraoperative navigation screenshots accurately predicted pedicle screw placement in 90.9% of cases. There was a positive correlation between 3D NAV accuracy and better screw grade (rs 0.45, p=0.036). CONCLUSIONS: Utilization of 3D NAV for percutaneous or mini-open lumbar screw placement during the early learning curve for MISS was associated with higher screw accuracy. This study demonstrates that the use of 3D navigation can facilitate the learning curve for MISS.

Neuronavigation in the minimally invasive presacral approach for lumbosacral fusion.

INTRODUCTION: Intraoperative 3D navigation (3D NAV) is gaining importance in spinal surgery, especially with the advancement of minimally invasive techniques in this field. We hypothesized that 3D NAV may be of benefit in the recently described minimally invasive presacral approach for L4-S1 fusion (AxiaLIF). METHODS: Five patients with refractory low back pain and L5-S1 degenerative disease underwent 3D NAV-assisted placement of a presacral L5-S1 screw. Additionally, a patient recently underwent a two-level AxiaLIF procedure (L4-S1) which was also performed with 3D NAV. Navigation was performed by acquiring intraoperative images via isocentric fluoroscopy and utilizing frameless stereotaxy. Accuracy of 3D NAV was confirmed by overlapping intraoperative images, navigation screenshots, and postoperative CT scans. Outcome was determined by pre- and postoperative VAS (back and leg pain) and ODI. RESULTS: Operative results were reviewed for all six patients and clinical outcomes were retrospectively reviewed for the five L5-S1 procedure patients. Postoperative imaging demonstrated accurate screw placement in comparison to intraoperative C-arm imaging and navigation imaging in all patients. No perioperative complications were encountered. In the five one-level patients, mean VAS (for back and leg pain) and ODI improved significantly at most recent follow-up. CONCLUSION: The minimally invasive presacral approach to L4-L5-S1 fusion can be performed safely and accurately with intraoperative 3D NAV. This is especially the case in two-level AxiaLIF procedures, where computer guidance can provide better planning possibilities for optimal screw trajectory.

Heparin and EDTA as anticoagulant differentially affect cytokine mRNA level of cultured porcine blood cells.

Cytokine mRNA expression profiles serve to characterize immune cell activation in different test systems. Both, diluted whole blood and isolated PBMC are widely applied for these studies. Comprehensive data regarding the suitability of different anticoagulants for profiling cytokine expression are not available for the pig. Therefore the aim of this study was to compare the effect of two commonly used anticoagulants (heparin and EDTA) on the cytokine expression pattern of porcine blood cells. IL-1alpha, IL-2, IL-4, IL-6, IL-10 and IFN-gamma mRNA levels were detected ex-vivo and upon in-vitro stimulation in diluted porcine whole blood and isolated PBMC by real-time PCR. The cells were stimulated with ConA or LPS, known to act on different target cells and implying different signalling pathways. Additionally the integrity of the isolated RNA was investigated. Ex-vivo cytokine expression pattern of fresh whole blood were not affected by the investigated anticoagulants. In contrast, stimulation of cultured diluted whole blood or PBMC resulted in significant differences depending on the applied anticoagulant. Using EDTA we found a significantly decreased capacity of whole blood to express cytokines. However, isolated PBMC from EDTA anticoagulated blood showed a higher cytokine expression capacity than PBMC from heparinized blood. Comparing diluted whole blood and PBMC we found that cultured porcine whole blood responded better to bacterial products than isolated PBMC, probably because sufficient auxiliary plasma derived factors such as LPS-binding protein, are present. However, isolated PBMC showed a higher T-cell response than diluted whole blood. In conclusion, our findings underline that each application demands a specific assay system.

Clenbuterol affects the expression of messenger RNA for interleukin 10 in peripheral leukocytes from horses challenged intrabronchially with lipopolysaccharides.

On four occasions, four horses with heaves and four horses with small airway inflammatory diseases inhaled 0.9% saline based aerosol mixtures with or without lipopolysaccharides (LPS). Prior to the first saline and LPS inhalation, horses were untreated, while three and a half days prior to the third and forth inhalation horses had received 0.8 microg/kg clenbuterol intravenously twice daily. The messenger RNA (mRNA) expression of tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-4, IL-6, IL-8, IL-10 and interferon- gamma (IFN- gamma) was investigated by RT-PCR, all of which were expressed in the white blood cells of samples collected. Inhalation of LPS only changed the cytokine expression profile of IL-10, IL-4 and TNF-alpha mRNA which were higher after challenge with LPS. However in those horses that were treated with clenbuterol the LPS-induced IL-10 mRNA expression was shown to be suppressed. Further changes in IL-4 and TNF-alpha were not significant. Thus the results of this study indicated that clenbuterol can modulate the expression of IL-10 mRNA in peripheral white blood cells in those horses with small airway diseases that have been exposed to LPS.

Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines.

We have established an easy real-time PCR assay, which allows the precise quantification of changes in the expression level of 6 relevant porcine cytokines, and 3 housekeeping genes. This assay simultaneously detects 9 sequences by measuring 3 x 3 targets in a triplex-format. The mRNA of the lymphokines IL-2, IL-4, IL-10, and IFN-gamma, of the proinflammatory cytokines IL-1alpha and IL-6, and of the housekeeping genes are quantified using TaqMan-probes by means of standard dilution series on the iCycler iQ. The standard consists of equal aliquots of the experimental cDNAs under investigation. Simultaneously the most suitable combination of 3 out of the four housekeeping genes beta-actin, HPRT, GAPDH, and cyclophilin can be selected, and their averaged expression values constitute a normalisation factor. The raw data of all targets of interest is then calculated relative to this normalisation factor, making eventual changes of the relative expression level of the single housekeeping genes controllable and quantifiable. We have applied this assay to quantify changes in the cytokine mRNA levels of porcine stimulated with various concentrations of LPS and ConA, known to induce different cytokine expression patterns. We have shown, that even small differences in the expression level (less than 2-fold) can be precisely quantified, and reveal statistically significant changes, when using the normalisation factor. This assay will be useful for studying changes in the expression of relevant porcine cytokines and will help to further improve the investigation of immune responses in the pig.

Characterization of the cytokine pattern of porcine bone marrow-derived cells treated with 1alpha,25(OH)D.

The biologically active form of vitamine D(3) [1alpha,25(OH)(2)D(3)] has recently been described not only to influence bone metabolism but also to exert immunomodulating activities, which may have an impact on bone formation/resorption as well. In this study, we analysed the effects of 1alpha,25(OH)(2)D(3) on the cytokine pattern of porcine bone marrow-derived cells from piglets aged 1-3 weeks. After culture for 1 week, the number of osteoclasts was determined, with tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated cells being considered osteoclasts. Cultured bone marrow cell-derived mRNA was subjected to semiquantitative RT-PCR specific for a panel of porcine cytokines (IL-1alpha, IL-6, IL-8, IL-10, and TNF-alpha). In addition, an immunofluorescence analysis using anti-porcine mAbs specific for IL-1beta, IL-2, IL-4, IL-6, IL-12, TNF-alpha, and IFN-gamma was performed. In order to prove the existence of a porcine homologue of the receptor activator of NF-kappaB ligand (RANKL) bone marrow cell- as well as porcine white blood cell-derived mRNA was investigated by RT-PCR using primer pairs specific for murine RANKL. Cell culture supernatant was analysed for soluble RANKL by means of an ELISA designed for quantification of human RANKL. By means of RT-PCR, expression of IL-1alpha, IL-6, IL-8, IL-10 and TNF-alpha mRNA could be found in cells cultured with and without 1alpha,25(OH)(2)D(3). Immunofluorescence analysis revealed that IL-1, IL-6, and TNF-alpha were produced by both stromal cells and osteoclasts. Besides its known osteoclastogenic effects, 1alpha,25(OH)(2)D(3) tended to downregulate the respective cytokines, but significantly upregulated RANKL expression. The homology between the porcine RANKL-specific sequence and the corresponding human RANKL sequence was 79%. The data found support the idea that porcine bone marrow cell cultures may provide a suitable alternative to murine systems in human osteological research.

Porcine dermatitis and nephropathy syndrome (PDNS) is associated with a systemic cytokine expression profile indicative of proinflammation and a Th1 bias.

Porcine dermatitis and nephropathy syndrome (PDNS) is broadly discussed as a porcine circovirus type 2 (PCV2)-associated disease, although PCV2, in contrast to postweaning multisystemic wasting syndrome (PMWS), has to date not been proven to be the aetiologic agent. In order to better understand the complex immunopathology of PDNS, the systemic cytokine expression profiles of (i) five pigs suffering from PDNS, (ii) five animals suffering from naturally acquired PMWS and (iii) five controls were investigated at mRNA and protein levels by means of multiplex real-time RT-PCR and flow cytometric intracellular cytokine detection, respectively. IL-1alpha, IL-6 and IFN-gamma mRNA expressions were found to be elevated in PDNS pigs. At the protein level, an increased capacity of peripheral blood mononuclear cells to produce IL-2, IL-4, IL-6, IL-12, TNF-alpha and IFN-gamma was evident. Hematological investigations revealed a hypochromic anemia while basophils and monocytes were relatively and neutrophils absolutely increased in PDNS pigs. PCV2 antibody levels did not differ significantly between PDNS and PMWS affected animals. Taken results together, the cytokine profile of the PDNS affected animals together with hematological data pointed towards a proinflammatory condition supporting a Th1 bias. Cytokine data of PMWS affected animals exhibited only minor non-significant differences when compared to controls, only IL-10 was significantly decreased at the mRNA level.

Systemic cytokine profile in feeder pigs suffering from natural postweaning multisystemic wasting syndrome (PMWS) as determined by semiquantitative RT-PCR and flow cytometric intracellular cytokine detection.

Postweaning multisystemic wasting syndrome (PMWS) is an economically important disease in pigs caused by porcine circovirus type 2 (PCV2). Development of this disease is presumably associated with an impairment of the immune system. We, therefore, investigated the systemic expression of relevant cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, TNF-alpha, IFN-gamma) and IL-2Ralpha at mRNA (semiquantitative RT-PCR) and at protein level (flow cytometric intracellular cytokine detection after short-time stimulation of peripheral blood mononuclear cells) in 10 feeder pigs aged 14 weeks suffering from natural PMWS and in 10 clinically healthy pen-mates. Hematological examination revealed a significant (p < 0.001) relative lymphopenia in the diseased animals when compared to reference pigs. IL-1alpha and IL-10 mRNA levels were notably increased in the affected pigs, whereas IL-2 and IL-2Ralpha (CD25) mRNA levels tended to be down-regulated. IL-8, TNF-alpha and IFN-gamma mRNA expressions appeared to be slightly increased. Intracellular cytokine levels as measured by flow cytometry revealed an increase of IL-1beta, IL-2, and IL-6, whereas IL-12 and TNF-alpha expressions were not affected. IFN-gamma was slightly decreased in the diseased animals. In conclusion, despite the assumption, that the cellular immune response to PMWS as a virus-induced disease should be characterized by either a Th1 driven cytokine profile or a cytokine profile indicative of T cell immunosuppression, our results did not support that hypothesis. Nevertheless, data from intracellular cytokine detection suggest an even increased percentage of the remaining lymphocytes capable to produce IL-2 upon in vitro stimulation, which is in contrast to the slightly diminished IL-2 mRNA levels reflecting the in vivo situation at least at the mRNA level.

Delay in processing porcine whole blood affects cytokine expression.

Quantification of cytokine messenger ribonucleic acid (mRNA) in blood samples has become an important tool in the investigation of immune cell activation in a variety of clinical settings. It has been shown that the method of sample collection and processing influences the levels of several cytokine mRNAs. Therefore, it is generally accepted that blood samples for analysis of cytokine expression be processed as soon as possible and under standardised conditions. Since immediate sample processing is not always possible, we investigated the effect of different storage conditions (room temperature (Rt) and 4 degrees C) and storage times (1, 2, 4, 6 and 24 h) on the mRNA level of different cytokines (IL-1alpha, IL-2, IL-6, IL-8, IL-10, IFN-gamma), as well as the IL-2 receptor (IL-2R) in porcine whole blood samples (n=8). Quantification of cytokine expression was performed using simultaneous reverse transcription PCR (RT-PCR) combined with the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference. Our data demonstrate that delays in sample processing longer than 1 h result in significant changes of the mRNA levels of individual cytokines. Expression of the monokines IL-1alpha, IL-6 and IL-10 were increased by storage at both room temperature and 4 degrees C. Expression of IL-8 was increased only in the samples stored at room temperature, and expression of IFN-gamma was raised exclusively in the samples stored at 4 degrees C. We conclude that porcine blood samples should be processed within 2 h to prevent undesired stimulatory effects on the cytokine expression pattern. However, if only selected cytokines are investigated, the undesired effects of prolonged storage can be selectively suppressed by choosing the appropriate temperature of sample storage.