Francisco Arnalich-Montiel

Immunosuppressive Properties of Mesenchymal Stem Cells: Advances and Applications

Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, such as bone marrow, skeletal muscle, dental pulp, bone, umbilical cord and adipose tissue. MSCs are used in regenerative medicine mainly based on their capacity to differentiate into specific cell types and also as bioreactors of soluble factors that will promote tissue regeneration from the damaged tissue cellular progenitors. In addition to these regenerative properties, MSCs hold an immunoregulatory capacity, and elicit immunosuppressive effects in a number of situations. Not only are they immunoprivileged cells, due to the low expression of class II Major Histocompatibilty Complex (MHC-II) and costimulatory molecules in their cell surface, but they also interfere with different pathways of the immune response by means of direct cell-to-cell interactions and soluble factor secretion. In vitro, MSCs inhibit cell proliferation of T cells, B-cells, natural killer cells (NK) and dendritic cells (DC), producing what is known as division arrest anergy. Moreover, MSCs can stop a variety of immune cell functions: cytokine secretion and cytotoxicity of T and NK cells; B cell maturation and antibody secretion; DC maturation and activation; as well as antigen presentation. It is thought that MSCs need to be activated to exert their immunomodulation skills. In this scenario, an inflammatory environment seems to be necessary to promote their effect and some inflammation-related molecules such as tumor necrosis factor-α and interferon-γ might be implicated. It has been observed that MSCs recruit T-regulatory lymphocytes (Tregs) to both lymphoid organs and graft. There is great controversy concerning the mechanisms and molecules involved in the immunosuppressive effect of MSCs. Prostaglandin E2, transforming growth factor-β, interleukins- 6 and 10, human leukocyte antigen-G5, matrix metalloproteinases, indoleamine-2,3-dioxygenase and nitric oxide are all candidates under investigation. In vivo studies have shown many discrepancies regarding the immunomodulatory properties of MSCs. These studies have been designed to test the efficacy of MSC therapy in two different immune settings: the prevention or treatment of allograft rejection episodes, and the ability to suppress abnormal immune response in autoimmune and inflammatory diseases. Preclinical studies have been conducted in rodents, rabbits and baboon monkeys among others for bone marrow, skin, heart, and corneal transplantation, graft versus host disease, hepatic and renal failure, lung injury, multiple sclerosis, rheumatoid arthritis, diabetes and lupus diseases. Preliminary results from some of these studies have led to human clinical trials that are currently being carried out. These include treatment of autoimmune diseases such as Crohns disease, ulcerative colitis, multiple sclerosis and type 1 diabetes mellitus; prevention of allograft rejection and enhancement of the survival of bone marrow and kidney grafts; and treatment of resistant graft versus host disease. We will try to shed light on all these studies, and analyze why the results are so contradictory.

Visual Field Index Rate and Event-Based Glaucoma Progression Analysis: Comparison in a Glaucoma Population

Aims: The aim of the study was to compare event-based glaucoma progression analysis (GPA) I with new GPA II software and pattern deviation-based trend analyses (visual field index [VFI]) to detect progression in a glaucoma population. Methods: This was a retrospective study that included 90 eyes of 90 patients with a minimum of five reliable visual field tests and a follow-up period of at least 2 years. Results: Event-based GPA II detected progression in 16.7% of eyes in which trend analysis VFI failed. GPA detected progression 6.8 months earlier than VFI. GPA I and II showed excellent agreement (k = 0.94). Agreement between VFI and mean deviation (MD) linear analysis and with GPA criteria was k = 0.52 and k = 0.48, respectively. Mean rates of progression of MD and VFI were −0.41 dB and −1.30% annually, respectively (rho = 0.824; p<0.0001). Using VFI, mean follow-up time was 6.12 and 4.89 years (p = 0.004) and the mean number of visual field tests was 7.33 and 6.01 (p = 0.023) in eyes with and without progression, respectively. Conclusions: Event-based software GPA I and II had excellent agreement. Event analysis showed earlier and greater sensitivity for detecting progression than VFI analysis and both had only moderate agreement. Trend analysis VFI is likely to detect progression in patients with a greater number of visual field tests and a longer follow-up time. The VFI analysis seems to be more accurate than MD analysis for determining rate of progression.

Cup-to-disc ratio: agreement between slit-lamp indirect ophthalmoscopic estimation and stratus optical coherence tomography measurement

PurposeTo determine agreement between slit-lamp indirect ophthalmoscopy and Stratus optical coherence tomography (OCT) when assessing cup-to-disc ratios (CDRs).MethodsTwenty-five ocular hypertensive subjects and 56 patients with primary open-angle glaucoma were included. Estimation of vertical (VCDR) and horizontal (HCDR) cup-to-disc ratio with slit-lamp ophthalmoscopy was made by three glaucoma specialists along with OCT scanning of optic nerve head. Agreement between OCT and specialists was measured by intraclass correlation coefficients (ICC), Bland and Altmans scatterplots, and a regression coefficient of the average difference.ResultsThe mean VCDR and HCDR was significantly higher (P<0.001) with OCT than that estimated by the specialists, with the difference ranging from 0.08 to 0.11, and from 0.13 to 0.18, respectively, depending on the specialist. Difference was higher (P<0.001) for cuppings below 0.3, and looses significance for larger VCDR cuppings (above 0.7). ICC for VCDR was 0.87 among specialists, and ranges from 0.82 to 0.75 when comparing OCT and specialists. ICC for HCDR was 0.83 among specialists and 0.74 between OCT and specialists. When data were plotted according to the Bland–Altman method, as the cupping increased, the agreement also increased.ConclusionsThere is very good agreement among the specialists when estimating CDRs by stereoscopic slit-lamp biomicroscopy. OCT shows higher values than the specialists; the greatest differences occurred when assessing small CDRs and the differences diminished as the cupping increased. These two methods of measurement are not interchangeable, and the difference must be considered, especially in discs with smaller CDRs.

Evaluation of the in vitro activity of commercially available moxifloxacin and voriconazole eye-drops against clinical strains of Acanthamoeba

PurposeAcanthamoeba is an opportunistic pathogen which is the causal agent of a sight-threatening ulceration of the cornea known as “Acanthamoeba keratitis” (AK) and, more rarely, an infection of the central nervous system called “granulomatous amoebic encephalitis” (GAE). The symptoms of AK are non-specific, and so it can be misdiagnosed as a viral, bacterial, or fungal keratitis. Furthermore, current therapeutic measures against AK are arduous, and show limited efficacy against the cyst stage of Acanthamoeba. Moxifloxacin, a fourth generation fluoroquinolone, has been used with other drugs to treat GAE, but its efficacy as a treatment for AK is not known. Voriconazole has been used to treat AK; however, its cysticidal efficacy is not known. Both drugs are commercially available as eye-drops. The aim of this study was to evaluate the in-vitro activity of these eye-drops against Acanthamoeba compared to two reference drugs (chlorhexidine and amphotericin B) which are currently used to treat AK and GAE.MethodsThe sensitivity of two clinical and one type strain of Acanthamoeba to the commercial concentrations of the four drugs was evaluated with a colorimetric assay. Mature cysts were incubated with voriconazole to determine their sensitivity to this drug. The effects on cell proliferation and cell toxicity were determined using standard procedures with commercial kits.ResultsThe four compounds were active against the Acanthamoeba strains in this study. Although it prevented encystation, moxifloxacin’s amoebicidal activity was low. Voriconazole activity was greater than that of the other drugs, even at a concentration lower than in commercial eye drops. It was effective against cysts and decreased cell proliferation, with low cellular cytotoxicity.ConclusionVoriconazole could be used against AK as a first-line treatment or in combination. Moxifloxacin is an interesting adjuvant to consider as it is effectively prevents encystation of the amoeba which often complicates infection resolution. In addition, moxifloxacin is effective in preventing secondary bacterial infections.

Acanthamoeba keratitis due to genotype T11 in a rigid gas permeable contact lens wearer in Spain

A case of a 59-year-old Spanish patient who presented with severe ocular pain, blurred vision, eyelid swelling and foreign body sensation in the right eye is reported. She was a regular gas permeable contact lens [corrected] wearer who initially claimed to maintain standard lens care. After exploration, conjunctival injection, dendritiform corneal ulcers and stromal edema were observed. She was initially treated for a possible viral keratitis due to herpes simplex virus using 3% topical acyclovir and 0.1% dexamethasone eye drops 5 times a day. The patient did not respond to this treatment and six weeks later, corneal scrapings were positive for Acanthamoeba genotype T11. She was then treated with chlorhexidine 0.02%, propamidine 0.1% and 1% cycloplegic eye drops hourly which resulted in a significant improvement. After a month, ocular pain decreased and the clinical signs of keratitis ameliorated observed as a diminution of the size of the ulcer and also in the extension and opacity of the corneal infiltrates. The patient has been following this treatment for 3 months and it is possible that she will have to carry on with it for a whole year. To the best of our knowledge, this is the first case of severe keratitis due to Acanthamoeba genotype T11 in Spain .

Optic disc cupping after optic neuritis evaluated with optic coherence tomography

PurposeTo determine whether the optic disc experiences cupping after an episode of optic neuritis as assessed by optical coherence tomography (OCT).MethodsA total of 50 patients with unilateral optic neuritis and 50 age- and sex-matched controls were studied. A complete examination, including visual acuity (VA), visual fields, and OCT scanning of the optic nerve head was performed. Mean cup-to-disc (C/D) ratios in the affected eyes were compared with fellow and control eyes.ResultsMean C/D area ratio (CDAR), C/D vertical ratio (CDVR), and C/D horizontal ratio (CDHR) were significantly larger in the affected eyes compared to fellow (P<0.001) and control eyes (P<0.05). The asymmetry in CDAR, CDVR, and CDHR between both eyes in the patients with optic neuritis was equal to or greater than 0.2 in 24, 28, and 30% respectively. A significant inverse correlation was found between the C/D ratios asymmetry and retinal nerve fibre layer (RNFL) thickness (P<0.05).ConclusionA significant increase in C/D ratio can be detected by OCT after unilateral optic neuritis, inversely correlated with RNFL thickness, and VA.

Mixed Acanthamoeba and multidrug-resistant Achromobacter xyloxidans in late-onset keratitis after laser in situ keratomileusis

A 31-year-old woman developed a spontaneous flap interface keratitis in the left eye 6 years after a laser in situ keratomileusis (LASIK) enhancement. Cultures and polymerase chain reaction (PCR) were positive for Achromobacter xyloxidans resistant to first- and second-generation cephalosporin, aminoglycosides, and quinolones and also positive for Acanthamoeba T4. Treatment with topical fortified ceftazidime, topical chlorhexidine, and voriconazole and oral voriconazole did not stop the progression of the disease. Flap amputation revealed persistence of Acanthamoeba but not Achromobacter. Six weeks after flap amputation, the infiltrate had resolved, PCR was negative for Acanthamoeba, and the cornea had fully epithelialized. To our knowledge, this is the first report of post-LASIK infectious keratitis caused by mixed infection of Achromobacter xyloxidans and Acanthamoeba occurring years after the procedure without apparent ocular trauma. It is also the first report of the use of combined systemic and topical voriconazole as a therapy for Acanthamoeba keratitis after LASIK.

Biointegration of corneal macroporous membranes based on poly(ethyl acrylate) copolymers in an experimental animal model.

Currently available keratoprosthesis models (nonbiological corneal substitutes) have a less than 75% graft survival rate at 2 years. We aimed at developing a model for keratoprosthesis based on the use of poly(ethyl acrylate) (PEA)-based copolymers, extracellular matrix-protein coating and colonization with adipose-derived mesenchymal stem cells. Human adipose tissue derived mesenchymal stem cells (h-ADASC) colonization efficiency of seven PEA-based copolymers in combination with four extracellular matrix coatings were evaluated in vitro. Then, macroporous membranes composed of the optimal PEA subtypes and coating proteins were implanted inside rabbit cornea. After a 3-month follow-up, the animals were euthanized, and the clinical and histological biointegration of the implanted material were assessed. h-ADASC adhered and survived when cultured in all PEA-based macroporous membranes. The addition of high hydrophilicity to PEA membranes decreased h-ADASC colonization in vitro. PEA-based copolymer containing 10% hydroxyethyl acrylate (PEA-HEA10) or 10% acrylic acid (PEA-AAc10) monomeric units showed the best cellular colonization rates. Collagen plus keratan sulfate-coated polymers demonstrated enhanced cellular colonization respect to fibronectin, collagen, or uncoated PEAs. In vivo implantation of membranes resulted in an extrusion rate of 72% for PEA, 50% for PEA-AAc10, but remarkably of 0% for PEA-HEA10. h-ADASC survival was demonstrated in all the membranes after 3 months follow-up. A slight reduction in the extrusion rate of h-ADASC colonized materials was observed. No significant differences between the groups with and without h-ADASC were detected respect to transparency or neovascularization. We propose PEA with low hydroxylation as a scaffold for the anchoring ring of future keratoprosthesis.

Adipose-derived mesenchymal stem cell administration does not improve corneal graft survival outcome.

The effect of local and systemic injections of mesenchymal stem cells derived from adipose tissue (AD-MSC) into rabbit models of corneal allograft rejection with either normal-risk or high-risk vascularized corneal beds was investigated. The models we present in this study are more similar to human corneal transplants than previously reported murine models. Our aim was to prevent transplant rejection and increase the length of graft survival. In the normal-risk transplant model, in contrast to our expectations, the injection of AD-MSC into the graft junction during surgery resulted in the induction of increased signs of inflammation such as corneal edema with increased thickness, and a higher level of infiltration of leukocytes. This process led to a lower survival of the graft compared with the sham-treated corneal transplants. In the high-risk transplant model, in which immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery, we found the use of systemic rabbit AD-MSCs prior to surgery, during surgery, and at various time points after surgery resulted in a shorter survival of the graft compared with the non-treated corneal grafts. Based on our results, local or systemic treatment with AD-MSCs to prevent corneal rejection in rabbit corneal models at normal or high risk of rejection does not increase survival but rather can increase inflammation and neovascularization and break the innate ocular immune privilege. This result can be partially explained by the immunomarkers, lack of immunosuppressive ability and immunophenotypical secretion molecules characterization of AD-MSC used in this study. Parameters including the risk of rejection, the inflammatory/vascularization environment, the cell source, the time of injection, the immunosuppression, the number of cells, and the mode of delivery must be established before translating the possible benefits of the use of MSCs in corneal transplants to clinical practice.

Co-isolation of Vahlkampfia and acanthamoeba in acanthamoeba-like keratitis in a Spanish population.

Purpose: To report the co-isolation incidence of Acanthamoeba and Vahlkampfia in amoebic keratitis from a tertiary care institution in Madrid, Spain. Methods: In this retrospective case series, 7 eyes of 7 consecutive patients with culture-proven or polymerase chain reaction–positive Acanthamoeba keratitis were seen at a tertiary care institution from January 2010 to April 2011, and their charts were reviewed. Results: Two of 7 patients showed mixed Acanthamoeba and Vahlkampfia keratitis. Good clinical response to the treatment was strongly correlated with early diagnosis, whereas delayed diagnosis resulted in poor response to the treatment in single or mixed infection. Conclusions: Co-isolation of Vahlkampfia and Acanthamoeba in Acanthamoeba-like keratitis has recently been detected in our population. This finding should raise awareness of the existence of other amoeba different from Acanthamoeba causing keratitis. There are not enough cases yet to determine the impact of mixed amoebic keratitis in the prognosis of this disease.