Combining Umbilical Cord Cells with Hyaluronic Acid Improves Heart Repair After a Heart Attack

Umbilical cord blood cells have an advantage over bone marrow or peripheral blood cells in that aging, systemic inflammation, and stress or damage caused by cell processing procedures can potentially compromise and diminish the regenerative capability of these cells. This problem is particularly acute in the case of treating patients who have recently suffered a heart attack, since transplanted cells experience a rather hostile environment that kills off most cells. Additionally, blood flow through the heart tends to wash out infused cells, which further decreases any regenerative activities the cells might have otherwise exerted.

With this in mind, Patrick Hsieh and his colleagues at the Academia Sinica, in Taipei, Taiwan tested if ability of human cord blood mononuclear cells (CB-MNCs) injected into the heart in combination with a hyaluronan (HA) hydrogel could extend the regenerative abilities of these cells in a pig model. HA is a common component of connective tissue, and, in general, it is very well tolerated by patients and implanted cells. Furthermore, it has the added bonus of shielding cells from a hostile environment and preventing them from being washed out of the heart.

Hsieh used a total of 34 minipigs and divided them into five different groups. One group was the sham operation group in which minipigs received surgical incisions but no heart attack was induced. The second group had heart attacks surgically induced and received infusions of normal saline solutions. The third group of minipigs also experienced heart attacks, and had HA injected into the heart walls. The fourth group also suffered heart attacks and received injections of human umbilical cord stem cells into their heart walls. The fifth group experienced heart attacks and received injections of both HA and human umbilical cord blood cells. The animals were kept and examined two months after surgery.

Two months after the surgery, the minipigs that received injections of human umbilical cord blood cells plus HA showed the highest left ventricle ejection fraction (51.32% ± 0.81%). This is significant when compared to 42.87% ± 0.97%, for the group that received injections of normal saline, 44.2% ± 0.63% for the group that received injections of HA alone, and 46.17% ± 0.39% for the group that received injections of umbilical cord blood cells only. Additionally, hearts from minipigs that received cord blood cells plus HA improved the systolic and diastolic function significantly better than the other experimental groups. Injections of either cord blood cells alone or in combination with HA significantly decreased the scar area and promoted the formation of new blood vessels in the infarcted region. In general, this study suggests that combined infusion of umbilical cord blood cells and HA improves the function of the heart after a heart attack and might prove to be a promising treatment option of heart attack patients.

This is a preclinical study, but it is a preclinical study in a larger animal model system. Umbilical cord blood cells have a demonstrated ability to induce healing in the heart after a heart attack. However, the combination of these cells with HA almost certainly significantly increases cell retention in the heart, thereby significantly improving cardiac performance, and preventing cardiac remodeling. Therefore, using healthy cells donated from another source to replace damaged or moribund cells may be a better option to treat a heart patient and repair their sick heart.

This work appeared in Stem Cells Trans Med November 2015, doi: 10.5966/sctm.2015-0092

Umbilical Cord Stem Cells Improve Heart Function after a Heart Attack

The umbilical cord connects the baby to the placenta and contains umbilical arteries, umbilical veins, and a gooey material between the umbilical vessels called Warton’s jelly. Warton’s Jelly (WJ), besides being rich in extracellular matrix molecules also contains a mesenchymal stem cell population that is rather primitive. These WJ mesenchymal stem cells or WJMSCs have excellent potential for therapeutic strategies.

Lian Gao and her colleagues from the Navy General Hospital in Beijing, China, in collaboration with coworkers from the Shenzhen Beike Cell Engineering Research Institute in Shenzhen, China conducted a clinical trial that examined the use of these WJMSCs in human patients who had suffered a heart attack.  The results are as interesting as they are suggestive and were published in the journal BMC Medicine.

First we must consider the design of the study. Gao and others recruited 160 heart attack patients who were no younger than 18 and no older than 80-years old. All patients had to be free of liver or kidney disease, cancer or some other terminal illness. They were admitted to 11 hospitals in China between February 2011 and January 2012 and had suffered from a documented heart attack as defined by symptoms and their EKG (ST elevation). All patients has also been treated with the implantation of a stent within 12 hours of their heart attack and still retained a respectable amount of movement of the heart wall in the left ventricle. If patients were outside these parameters, they were excluded from the study.

Of the 160 patients who were recruited for the study, 44 were excluded, either because they did not fit within the exclusion criteria, did not wish to participate in the trial, or opted out for undisclosed reasons. This left 116 patients who were randomly assigned to the placebo group or the experimental group (58 in each group). Of these two groups, the placebo group had one patient discontinue the study because of a bout with stomach cancer. The experimental group had one patient die ten days into the trial, another was lost because they moved and a third patients withdrew because of leukemia. This left 57 subjects for the placebo group and 55 for the experiment group who went through all 18 months of follow-up after their respective procedures.

There were two end points for this clinical trial after patients were observed for 18 months after the procedure. The first was safety and this was measured by examining the number of adverse effects (AEs) within these 18 months. Such AEs include things like death, hospitalization for worsening heart function, severe arrhythmias, repeated coronary intervention, blood clots forming in the stents (stent thrombosis), coronary artery obstruction, and the growth of extra tissue in the heart that does not belong there, disorders of the immune system and so on. The second end pointy was efficacy of the implanted cells. To ascertain this, the function of the heart was measured using positron emission computer tomography (PET), and single-photo-emission computer tomography or SPECT. These imaging procedures allow cardiologists to take very precise snapshots of the heart and determine with a good deal of accuracy the performance of the heart.

The WJMSCs were acquired from umbilical cords that were donated from healthy mothers who had delivered healthy babies by means of Caesarian section. 21 of these umbilical cords had their blood vessels removed and then the gelatinous tissue surrounding the vessels was removed, sliced up, and cultured. The MSCs in the gelatinous tissue, which is Warton’s Jelly, migrated from the WJ to the culture dishes. After three passages in the culture dishes, he cells were harvested, concentrated, and tested for viruses, toxins, and cell viability. All cells were negative for viruses and toxins and other contaminants, and were also clearly MSCs, based on the ensemble of cell surface proteins that presented on their membranes, and showed high degrees of viability.

In infuse the cells into the hearts of the patients, six million WJMSCs were delivered into the coronary arteries using the usual over-the-wire techniques that are used to place stents, except that instead of placing stents, WJMSCs were slowly released into the coronary arteries. The cells will home to the damaged heart tissue and are able to pass through the blood vessels into the area of the infarct. Patients receiving the placebo, only received infusions of physiological saline solution, which was used to resuspend the WJMSCs.

The results are very encouraging. With respect to safety, the number of AEs was approximately the same for both groups. In the words of the study, “The groups did not differ in occurrences of MACEs (major adverse cardiac events), including death, recurrences of AMIs (acute myocardial infarctions) and re-hospitalization due to heart failure, during the course of treatment and the 18-month follow-up period.” There were no indications of cancer or the increase in tumor-specific molecules in the blood of the patients from either group. No biochemical or immune abnormalities were observed in any pf the patients either. The stomach cancer in one patient in the placebo group and leukemia in a patient from the experimental group were shown to be unrelated to the procedures. Therefore, at 18 months after the procedure, the infusion of these cells appears to be safe.

As to the efficacy of the procedure, there were significant improvements in the heart function of patients who had received the WJMSCs over those who had received placebo. First of all, the baseline heart function of patients in both groups was approximately the same on the average, except that the patients in the experimental group had slightly better heart parameter than those in the placebo group. Therefore, the efficacy of this procedure was determined by measuring the change in heart performance after the procedure. Patients who had received the placebo had about a 3% increase in the uptake of the F18-labeled sugar molecule after 4 months. The uptake of this marker indicates the presence of live cells. An increase in uptake of the modified sugar molecule shows that some new heart tissue has been produced, probably by the resident stem cell population in the heart. The experimental group, however, after 4 months showed an approximate 7% increase in PET signal intensity. This shows that a good deal more heart cells are being formed in the WJMSC-treated hearts that in the placebo-treated hearts. The SPECT imaging assays the “perfusion” of the heart tissue or the degree to which the heart tissue is being fed by blood vessels. After a heart attack, the dead area of the heart lacks blood vessels and its poor perfusion can affect nearby areas. The placebo-treated patients had a roughly 4% increase in SPECT signal, whereas the WJMSC-treated group had a 7% increase. Thus, the WJMSC-treated hearts had more blood vessels to feed the blood, oxygen and nutrients to the heart muscle and therefore, better perfusion.

Finally, the percentage of blood ejected by the heart during each contraction increase about 3% in the placebo group, but increase by about 8% in the WJMSC-treated group after 18 months. This parameter of heart function, the ejection fraction, is a very important measure of heart function and the fact that it significantly increased in the WJMSC-treated patients over the placebo-treated patients is an important finding.

This was a double-blinded, placebo-controlled study that determined the safety and efficacy of infusions of WJMSCs into the hearts of patients who had recently suffered from a heart attack. In animal experiments, these cells have been shown to increase heart function, increase blood vessel density in the hearts of animals, and increase resident heart-specific stem cell activity in the heart (see Lupu and others, Cell Physiol Biochem 2011; 28:63-76; Gao and others, Cell Transplant 2013; 22:1883-1900; Lopez Y, and others, Current Stem Cell Res Ther 2013;8:46-59). This clinical trial suggests that those benefits documented in laboratory animals might translate to human patients.

This is not a perfect study. These patients will need to be followed for several years to establish that these benefits are long-term and not short-term. Also, there is no indication that patients were given a 6-minute walking test to determine if the improvements in cardiac function translated to improvements in basic activities. However, it is an interesting study and it suggests that banking WJMSCs in addition to cord blood might be a good idea for use in trials like this one and maybe, someday for treatments of heart attack patients.

Culture Medium from Human Amniotic Membrane Mesenchymal Stem Cells Promotes Cell Survival and Blood Vessel Production in Damaged Rat Hearts

The laboratory of Massimiliono Gnecchi at the Fondazione IRCCS Policlinico San Matteo in Pavia, Italy has used the products of amniotic mesenchymal stem cells to treat heart attacks in laboratory rodents. The results are rather interesting.

In a paper published in the May 2015 edition of the journal Stem Cells Translational Medicine, Gnecchi and his colleagues grew human amniotic mesenchymal stem cells derived from amniotic membrane (hAMCs) in cell culture.

These cells were isolated from amniotic membrane donated by mothers who were undergoing Caesarian sections. The membranes were removed, and grown in standard culture media under standard conditions. Once the cells grew out, they were collected and grow in a medium known as DMEM (Dulbecco’s modified Eagle Medium). After the cells had grown for 36 hours, they culture medium was filtered, concentrated, and readied for use.

The first experiments included the use of this conditioned culture medium to treat H9c2 embryonic heart muscle cells with in culture and then expose the heart muscle cells to low oxygen conditions. Normally, low oxygen conditions kill heart muscle cells. However, the cells pre-treated with conditioned medium from hAMCs showed much more robust survival in low-oxygen conditions. This shows that molecules secreted by hAMCs had promote the survival of heart muscle cells.

Next, Gnecchi and his team used their conditioned medium to treat laboratory rats that had suffered heart attacks. Some of the rats were treated with conditioned culture medium from cultured skin cells and others with sterile saline. The culture medium was injected directly into the heart muscle.  The rats treated with conditioned medium from hAMCs showed far less cell death than the other rats. The rats treated with the hAMC-treated culture medium also had vastly denser concentrations of new blood vessels.

It is well-known that mesenchymal stem cells from many sources are filled with small vesicles known as exosomes that are loaded with healing molecules. Mesenchymal stem cells release these exosomes when they home to damaged tissues. The culture medium from the hAMCs were almost certainly filled with exosomes. The molecules released by these cells helped promote heart muscle cell survival in the oxygen-depleted heart, and induced the recruited large numbers of EPCs (endothelial progenitor cells), which established large numbers of new blood vessels. These new blood vessels gave oxygen to formerly depleted heart tissue and promoted heart healing. The size of the heart scar was smaller in the rats treated with hAMC-conditioned medium.

Unfortunately there were no measurement of cardiac function so we are not told if this treatment affected ejection fraction, or other physiological parameters. Nevertheless, this paper does show that exosomes from hAMCs do promote the production of blood vessels and cell survival.

Dead Heart Muscle Regrown in Rodents

If you cut a piece of tissue from the heart of a salamander or zebrafish, they wild simply grow new heart tissue. Unfortunately, humans are unable to easily regenerate heart cells, and this males it difficult to recover from the permanent damage caused by heart attacks.

Fortunately, life scientists from the Weizmann Institute of Science in Israel and the Victor Chang Institute in Sydney have discovered a way to stimulate heart muscle cells in mammals to grow. This finding could have major implications for future heart attack sufferers.

Even though human blood, hair and skin cells renew themselves throughout life, cell division in the heart comes to a virtual standstill shortly after birth, according to Prof. Richard Harvey, from the Victor Chang cardiac research institute, and one of the authors of this research. Harvey said, “So there’s always been an intense interest in the mechanism salamanders and fish use which makes them capable of heart regeneration, and one thing they do is send their cardiomyocytes, or muscle cells, into a dormant state, which they then come out of to go into a proliferative state, which means they start dividing rapidly and replacing lost cardiomyocytes.”

Harvey continued: “There are various theories why the human heart can not do that, one being that our more sophisticated immune system has come at a cost, and because human cardiomyocytes are in a deeper state of quiescence, that has made it very difficult to stimulate them to divide.”

Today, for the first time in history, more people in developing countries die from strokes and heart attacks than infectious diseases. Fortunately there are cost-effective ways to save lives

By studying mice, Harvey and his colleagues found a way to overcome that regenerative barrier – at least in the rodents.

Harvey and others found that by stimulating a cell signaling pathway in the heart that is driven by a hormone called neuregulin, heart muscle cells divided in a spectacular way in both adolescent and adult mice. In humans, neuregulin expression is usually muted about one week after birth, and by about 20 weeks after birth in mice.

By triggering of the neuregulin pathway following a heart attack in mice, Harvey and others induced the replacement of lost muscle, which repaired the heart to a level close to that prior to the heart attack. Harvey said that he and other scientists should be able to determine with in the next five years if it is possible to replicate these results in humans.

“This is such a significant finding that it will harness research activities in many labs around the world, and there will be much more attention now on how this neuregulin-response could be maximised,” Harvey said.

“We will now examine what else we can use, other than genes, to activate that pathway, and it could be that there are already drugs out there, used for other conditions and regarded as safe, that can trigger this response in humans.”

When one of the blood vessels that provide blood to the heart muscle becomes blocked, the patient suffers a heart attack. Heart attacks or “myocardial infractions” cause billions of cardiomyocytes to die. Even if you survive a heart attack, you usually experience diminished quality of life because of it.

“The dream is that one day we will be able to regenerate damaged heart tissue, much like a salamander can regrow a new limb if it is bitten off by a predator,” Harvey said.

Molecular biologist Gabriele D’Uva lead this research, which was published in the scientific journal Nature Cell Biology.

Nonhematopoietic Stem Cells from Umbilical Cord Blood Improve Heart Function After a Heart Attack

Xin Yu, who has dual appointments at Case Western Reserve University, in Cleveland, Ohio and the University of Minnesota Medical School in Minneapolis, Minnesota, has published a remarkable paper in the journal Cell Transplantation that describes the use of a stem cell population from umbilical cord blood to treat mice that had suffered heart attacks. The non-invasive way in which these cells were administered and the tremendous healing qualities of these cells makes paper unique.

In 2005, Water Low at the University of Minnesota Medical School described the isolation and characterization of a unique stem cell population from umbilical cord blood that he called nonhematopoietic umbilical cord blood stem cells or nh-UCBSCs. These cells were used to treat animals with strokes and they induced the growth of new brain cells in the brains of treated mice (see J Xiao, Z Nan, Y Motooka, and WC Low, Stem Cells Dev. 2005 Dec;14(6):722-33).

Yu used these cells to treat male Lewis rats that had suffered heart attacks. In all cases, the rats were subjected to open-heart surgery and the left anterior descending artery was tied off to induce a heart attack. One group of rats were operated on but no heart attacks were induced. A second group was given heart attacks, and then two days later were given intravenous saline infusions. The third group was given a heart attack and then two days later, were injected with one million nonhematopoietic umbilical cord stem cells into their tail veins.

Ten months after the surgery, the heart structure and function of animals from all three groups was assessed with tensor diffusion magnetic imaging, and a pressure‐volume conductance catheter. The hearts were also extirpated from the animals and structurally assessed by means of staining and 3-D imaging.

The stem cell-treated animals were compared with the sham-operated animals and the saline-treated animals. In almost all categories, the stem cell-treated animals had better function. Also, the overall structure of the heart was preserved and looked more like the normal heart than the saline-treated hearts. For example, in the saline-treated group, the heart wall thickness in the infarct zone was reduced by 50% compared to the control rats, and wall thickness at the border zone was also significantly
decreased. However, there were no statistical difference in wall thickness between the stem cell-treated group and the control group.

Additional finds were that the stem cell-treated group had significantly smaller areas of dead cells, more blood vessels, and better heart muscle fiber structure that contracted better.

These data show that the long-term effects of nh-UCBSC administration was to preserve the structure, and, consequently, the function of the heart after a heart attack.

However, the added bonus to this work is that the animals were injected with these cells into the tail vein. The animals did not have to have their chests cracked, or have over-the-wire stent technology to implant these cells; they merely introduced them intravenously. Apparently, the nh-UCBSCs homed to the damaged heart and mediated its healing. If such healing can be translated to human patients, this could truly be a revolutionary find.

Five-Year Follow-up of REPAIR-AMI Clinical Trial

The REPAIR-AMI clinical trial was a double-blind placebo-controlled trial in which 204 recent heart attach patients received either an infusion of bone marrow stem cells or a placebo. The results of this clinical trial have been published in three different papers (Schächinger, et al., N Engl J Med 2006 355: 1210-1221; Schächinger, et al., Eur Heart Journal 2006 27: 2775-2783; Schächinger, et al., Nat Clin Pract Cardvasc Med 2006 3(Suppl 1): 523-528).

This clinical trial showed that the bone marrow-treated group showed significant functional improvements over the placebo group. However, a long-term follow-up of these patients was required to demonstrate that the benefits conferred by the stem cell treatments were long-lasting and not merely transient.

Upon 5-year examination, the stem cell-treated group showed lower rates of a second heart attack, hospitalization, strokes, cancer, surgical interventions to open blocked vessels and death. Thus, the stem cell-treated group fared better in almost all the major categories.

There was, however, an additional experiment that gave a truly remarkable result. After each patient had their bone marrow extracted, the stem cells were subjected to individual tests, one of which were mobility tests. When this research group examined the stem cell motility data and correlated it to the five-year follow-up, they discovered a very tight association between the motility of the bone marrow stem cells and the absence of cardiac events. More active bone marrow cells provided greater recovery and fewer post-procedural events.

These data show that the quality of the bone marrow is a significant factor in the success of the stem cell treatment.

This also brings up another question: Can be beef up the quality of the bone marrow some how? Culturing stem cells can expand them, but it can also significantly change them. Therefore, this remains a fertile field for research and development, and the bone marrow quality may also explain why bone marrow transplants into the heart work so well or some patients and not at all for others.

New US Phase IIa Trial and Phase III Trial in Kazakhstan Examine CardioCell’s itMSC Therapy to Treat Heart Attack Patients

The regenerative medicine company CardioCell LLC has announced two new clinical trials in two different countries that utilize its allogeneic stem-cell therapy to treat subjects with acute myocardial infarction (AMI), which is a problem that faces more than 1.26 million Americans annually. The United States-based trial is a Phase IIa AMI clinical trial that is designed to evaluate the clinical safety and efficacy of the CardioCell Ischemia-Tolerant Mesenchymal Stem Cells or itMSCs. The second clinical trial in collaboration with the Ministry of Health in Kazakhstan is a Phase III AMI clinical trial on the intravenous administration of CardioCell’s itMSCs. This clinical trial is proceeding on the strength of the efficacy and safety of itMSCs showed in previous Phase II clinical trials.

CardioCell’s itMSCs are exclusively licensed from CardioCell’s parent company Stemedica Cell Technologies Inc. Normally, when mesenchymal stem cells from fat, bone marrow, or some other tissue source are grown in the laboratory, the cells are provided with normal concentrations of oxygen. However, CardioCell itMSCs are grown under low oxygen or hypoxic conditions. Such growth conditions more closely mimic the environment in which these stem cells normally live in the body. By growing these MSCs under these low-oxygen conditions, the cells become tolerant to low-oxygen conditions (ischemia-tolerant), and if transplanted into other low-oxygen environments, they will flourish rather than die.

Another advantage of itMSCs for regenerative treatments over other types of MSCs is that itMSCs secrete higher levels of growth factors that induce the formation of new blood vessels and promote tissue healing. These clinical trials have been designed to help determine if CardioCell’s itMSC-based therapies stimulate a regenerative response in acute heart attack patients.

“CardioCell’s new Phase IIa AMI study is built on the excellent safety data reported in previous Phase I clinical trials using our unique, hypoxically grown stem cells,” says Dr. Sergey Sikora, Ph.D., CardioCell’s president and CEO. “We are also pleased to report that the Ministry of Health in Kazakhstan is proceeding with a Phase III CardioCell-therapy study following its Phase II study that was highly promising in terms of efficacy and safety. Our studies target AMI patients who have depressed left ventricular ejection fraction (LVEF), which makes them prone to developing extensive scarring and therefore to the development of chronic heart failure. CardioCell hopes our itMSC therapies will inhibit the development of extensive scarring and, thus, the occurrence of chronic heart failure in these patients.”

The United States-based Phase IIa clinical trial will take place at Emory University, Sanford Health and Mercy Gilbert Medical Center. The CardioCell Phase IIa AMI trial is a double-blinded, multicenter, randomized study designed to assess the safety, tolerability and preliminary clinical efficacy of a single, intravenous dose of allogeneic mesenchymal bone-marrow cells infused into subjects with ST segment-elevation myocardial infarction (STEMI).

“While stem-cell therapy for cardiovascular disease is nothing new, CardioCell is bringing to the field a new, unique type of stem-cell technology that has the possibility of being more effective than other AMI treatments,” says MedStar Heart Institute’s Director of Translational and Vascular Biology Research and CardioCell’s Scientific Advisory Board Chair Dr. Stephen Epstein. “Evidence exists demonstrating that MSCs grown under hypoxic conditions express higher levels of molecules associated with angiogenesis and healing processes. There is also evidence indicating they migrate with greater avidity to various cytokines and growth factors and, most importantly, home more robustly to ischemic tissue. Studies like those underway using CardioCell’s technology are designed to determine if we can evoke a more potent healing response that will reduce the extent of myocardial cell death occurring during AMI and thereby decrease the amount of scar tissue resulting from the infarct. A therapy that could achieve this would have a major beneficial impact in reducing the occurrence of chronic heart failure.”

Kazakhstan’s National Scientific Medical Center is conducting a Phase III AMI clinical trial using CardioCell’s itMSCs, which are sponsored by local licensee Altaco. This clinical trial is entitled, “Intravenous Administration of itMSCs for AMI Patients,” and is proceeding based on a completed Phase II efficacy and safety study. However, the results of this previous Phase II study are preliminary because the sample group was so small. Despite these limitations, the findings demonstrated statistically significant elevation (more than 12 percent over the control group) in the ejection fraction of the left ventricle of the heart in patients who had received itMSCs. Also, a significant reduction in inflammation was also observed, as ascertained by lower CRP (C-reactive protein) levels in the blood of treated patients in comparison to control groups. Thus, Dr. Daniyar Jumaniyazov, M.D., Ph.D., principal investigator in Kazakhstan clinical trials states: “In our clinical Phase II trial for patients with AMI, treatment using itMSCs improved global and local myocardial function and normalized systolic and diastolic left ventricular filling, as compared to the control group. We are encouraged by these results and look forward to confirming them in a Phase III study.”

CardioCell’s treatment is the first to apply itMSC therapies for cardiovascular indications like AMI, chronic heart failure and peripheral artery disease. Manufactured by CardioCell’s parent company Stemedica and approved for use in clinical trials, itMSCs are manufactured under Stemedica’s patented, continuous-low-oxygen conditions and proprietary media, which provide itMSCs’ unique benefits: increased potency, safety and scalability. itMSCs differ from competing MSCs in two key areas. itMSCs demonstrate increased migratory ability towards the place of injury, and they show increased secretion of growth and transcription factors (e.g., VEGF, FGF and HIF-1), as demonstrated in a peer-reviewed publication (Vertelov et al., 2013). This can potentially lead to improved regenerative abilities of itMSCs. In addition, itMSCs have significantly fewer HLA-DR receptors on the cell surface than normal MSCs, which might reduce the propensity to cause immune responses. As another benefit, itMSCs are highly scalable. A single donor specimen can currently yield about 1 million patient treatments, and this number is expected to grow to 10 million once full robotization of Stemedica’s facility is complete.