Kathryn Shaw

Human mesenchymal stem cells attenuate early damage in a ventilated pig model of acute lung injury

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a major cause of global morbidity and mortality. Mesenchymal stem cells (MSC) have shown promise in treating inflammatory lung conditions. We hypothesised that human MSC (hMSC) can improve ALI/ARDS through their anti-inflammatory actions. We subjected pigs (n=6) to intravenous oleic acid (OA) injury, ventilation and hMSC infusion, while the controls (n=5) had intravenous OA, ventilation and an infusion vehicle control. hMSC were infused 1h after the administration of OA. The animals were monitored for additional 4h. Nuclear translocation of nuclear factor-light chain enhancer of activated B cells (NF-κB), a transcription factor that mediates several inflammatory pathways was reduced in hMSC treated pigs compared to controls (p=0.04). There was no significant difference in lung injury, assessed by histological scoring in hMSC treated pigs versus controls (p=0.063). There was no difference in neutrophil counts between hMSC-treated pigs and controls. Within 4h, there was no difference in the levels of IL-10 and IL-8 pre- and post-treatment with hMSC. In addition, there was no difference in hemodynamics, lung mechanics or arterial blood gases between hMSC treated animals and controls. Subsequent studies are required to determine if the observed decrease in inflammatory transcription factors will translate into improvement in inflammation and in physiological parameters over the long term.

Cranial reconstruction using allogeneic mesenchymal stromal cells: a phase 1 first-in-human trial

Cranioplasty is necessary for patients that have undergone craniectomy following trauma, stroke or other causes of elevated intracranial pressure. This study assessed the effectiveness of treating cranial defects with allogeneic mesenchymal stromal cells (MSC) on a ceramic carrier and polymer scaffold, to produce viable bone and healing of a cranial void. Patients underwent a baseline computed tomography (CT) scan for construct design. Two sets of interlocking moulds were three‐dimensional printed to enable shaping of two polymer meshes, which formed the boundaries of the construct corresponding to restoration of the skull interna and externa. In vitro expanded donor MSC were seeded onto ceramic granules in a good manufacturing practices facility. The inner mesh was placed in theatre, followed by the cell‐loaded granules, and the outer mesh. Patients were followed‐up at 3, 6 and 12 months and cosmesis assessed visually, while bone formation was assessed by CT scans at 1 day, 3 months and 12 months. Manufacture of the construct and surgery was uneventful for all three patients. Initial cosmesis was excellent with no complications. New bone formation was demonstrated by analysis of CT data; however, bone resorption was noted in all 3 cases on the 12‐month CT scan. The lack of rigidity of the construct in an environment with continuous pulsatile movement may be preventing the formation of solid bone. It is possible to produce a customized allogeneic MSC construct for cranial reconstruction to replace cranial bone with good cosmesis, using a combination of medical computer modelling, rapid‐prototyping and tissue engineering.

Mesenchymal stromal cell infusion modulates systemic immunological responses in stable COPD patients: a phase I pilot study

Chronic obstructive pulmonary disease (COPD) is a leading cause of global morbidity and mortality due to limited therapeutic options for the persistent pulmonary and systemic inflammation that characterises this condition [1]. Recently, pre-clinical studies of mesenchymal stromal cells (MSCs) in COPD demonstrate efficacy in alleviating inflammation and reducing emphysema following either systemic or intra-tracheal administration [2, 3]. Human trials have demonstrated that MSCs did not improve spirometry following their administration to COPD patients; however, it was reported that C-reactive protein (CRP), a marker for systemic inflammation, was reduced 1–3 months after infusion. Earlier time-points were not assessed in detail in these trials, which limits further investigation of these changes [4, 5]. Identifying the fate of intravenously infused MSCs and the potential implications of their biodistribution, as well as short-term MSC-induced systemic changes that were not explored in previous trials will better delineate the utility of MSC treatment for COPD. Mesenchymal stromal cell infusion may provide an alternative immune-based therapeutic option for COPD patients http://ow.ly/5DpA30hQS6y

Clinical significance of marrow micrometastases on outcome in operable non-small-cell lung cancer

The success of surgical resection for early stage non-small cell lung cancer (NSCLC) is largely determined by the presence of undetectable spread at the time of resection. Survival after resection of Stages I–IIIa NSCLC is limited by loco-regional or distant recurrence and it would be advantageous to have a technique, which identified patients who already had extrapulmonary metastasis at the time of surgery. This would potentially allow amore accurate prognosis, and separate a group which might benefit from adjuvant therapy. Passlick et al. reported a technique in 1999 which identified epithelial antigen in marrow elements, concluding that this represented true metastasis and was an independent risk factor for overall survival.1 We have applied a technique up to 100 times more sensitive than previously reported, being able to find as few as one tumour cell in 108 marrow elements. Thirty-eight patients with resectable NSCLC were enrolled in the study from February 1999 to September 2002. The study was censored in May 2003 after a cumulative 824 patient-months (range 2–50). After anaesthesia and before the chest incision, a 5–10mL sample of bone marrow was aspirated from the ipsilateral posterior superior iliac spine and placed in HEPES-buffered RPMI-1640 cell culture medium supplemented with 100U/mL heparin and chilled for transport to the laboratory. On receival, the samplewas diluted with an equal volume of RPMI/heparin (100U/mL), cells sedimented at 400 g for 10min, cell pellet resuspended in 10mL of RPMI containing DNase (100U/mL) and incubated with gentle rotation for 30min. The sample was then immunomagnetically enriched for epithelial tumour cells using the MACS Carcinoma CellEnrichment Kit (Miltenyi Biotec, Berdisch Gladbach, Germany) according to the manufacturer’s recommendations. Briefly, cells (0.1–3.4· 108, mean 1.42 – 0.14 (standard error of mean) · 108) were first permeabilized and fixed, then incubated with cytokeratin antibody (CAM 5.2, Beckton Dickinson, San Jose, CA, USA) attached to magnetic microbeads, which allows the magnetic selection of carcinoma cells. Labelled cells were then separated on a MACS RS+ separation column (Miltenyi Biotec) using a VarioMACS magnet (Miltenyi Biotec). Carcinoma cells were detected on cell cytocentrifuge preparations immunohistochemically, using cytokeratin antibodies (CAM 5.2, LP34 and MNF 116, Dako, Glostrup, Germany) recognizing cytokeratins 5, 6, 7, 8, 10, 17, 18 and 19, including appropriate controls. Cells were counterstained with haematoxylin and evaluated by light microscopy. Positive-staining cells were verified by morphology. At surgery, a sample was also taken from the resected NSCLC by TruCut biopsy and transported and assayed with the bone marrow sample to confirm immunohistochemical identification. All cancers were felt to be completely resected and there were no perioperative deaths or significant complications. Patients were followed clinically and radiologically at 3, 6, 12, 18 and 24months and yearly thereafter. Recurrence of tumour or the establishment of cause of death was assessed by review of public and private hospital case notes and databases, contact with patient Specialist, GP or family, obtaining copies of death certificates and searching the West Australian State Mortality and Cemetery databases. Patients were managed in the standard manner, without knowledge of the results of the assay. Of the 38 patients, 15 had bone marrow samples that tested positive for carcinoma cells (with similar immunohistochemical staining as their lung tumour) and 23 were negative. There was no significant difference in the number of cells analysed from the patients in each group. Of the 15 positive patients, 9 are alive and cancer free and 6 have died. Two died of recurrent bronchogenic tumour (17 and 36months postoperative), one died of new, metastatic rectal carcinoma (8months post-thoracotomy), one died of new pancreatic cancer (36months postthoracotomy) and two died in cardiogenic shock (2months postthoracotomy, 1week post-CABG; and 13months post-thoracotomy, from urinary sepsis). Therefore, 2 of 11 evaluable positive patients (18.2%) died of recurrent NSCLC with a mean follow up for this group of 25months (range, 8–50months). Of the 23 negative patients, 16 are alive with no evidence of recurrent tumour and 7 have died. Four of these seven patients died of recurrent NSCLC (19, 20, 22 and 24months from surgery). Of the remaining three, one died of pancreatic carcinoma (4months postthoracotomy), one died of renal failure with pneumonia (3months post-thoracotomy) and one of multiple pulmonary emboli (24months post-thoracotomy). Therefore, 4 of 20 evaluable negative patients (20%) died of recurrent NSCLC with a mean follow up for this group of 22 months (range, 8–46months). It is clear that this limited study cannot support the hypothesis that the presence of preoperative marrow micrometastasis in resectable NSCLC identifies a higher risk group for recurrence of tumour. Therefore, the test as described has no clinical usefulness. Concurrent reported work confirms this.2–4 Patient numbers are small and no attempt was made to assess statistical significances. The follow-up period is relatively short, but in view of the high number of negative marrow patients succumbing to recurrent NSCLC within 24months, it was felt that continued observation was not warranted.