The effect of Me2SO overexposure during cryopreservation on HOS TE85 and hMSC viability, growth and quality.

With the cell therapy industry continuing to grow, the ability to preserve clinical grade cells, including mesenchymal stem cells (MSCs), whilst retaining cell viability and function remains critical for the generation of off-the-shelf therapies. Cryopreservation of MSCs, using slow freezing, is an established process at lab scale. However, the cytotoxicity of cryoprotectants, like Me2SO, raises questions about the impact of prolonged cell exposure to cryoprotectant at temperatures >0 °C during processing of large cell batches for allogenic therapies prior to rapid cooling in a controlled rate freezer or in the clinic prior to administration. Here we show that exposure of human bone marrow derived MSCs to Me2SO for ≥1 h before freezing, or after thawing, degrades membrane integrity, short-term cell attachment efficiency and alters cell immunophenotype. After 2 h's exposure to Me2SO at 37 °C post-thaw, membrane integrity dropped to ∼70% and only ∼50% of cells retained the ability to adhere to tissue culture plastic. Furthermore, only 70% of the recovered MSCs retained an immunophenotype consistent with the ISCT minimal criteria after exposure. We also saw a similar loss of membrane integrity and attachment efficiency after exposing osteoblast (HOS TE85) cells to Me2SO before, and after, cryopreservation. Overall, these results show that freezing medium exposure is a critical determinant of product quality as process scale increases. Defining and reporting cell sensitivity to freezing medium exposure, both before and after cryopreservation, enables a fair judgement of how scalable a particular cryopreservation process can be, and consequently whether the therapy has commercial feasibility.

Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation.

For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me2SO). Me2SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me2SO. Although reduced (by 22 ± 2%, p=0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me2SO, where the doubling time was significantly reduced (by 41 ± 4%, p=0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.

Yeast Eps15-like endocytic protein, Pan1p, activates the Arp2/3 complex.

Longstanding evidence supports a role for actin in endocytosis; an intact actin cytoskeleton is required for endocytosis in yeast, and drugs that inhibit actin polymerization inhibit endocytosis in both yeast and mammalian cells. The yeast Arp2/3 complex is required for the internalization step of endocytosis. In addition, some early endocytic events in mammalian cells are associated with the formation of actin tails similar to those generated by activated Arp2/3 complex. However, until now no Arp2/3 complex activator has been identified among proteins known to mediate early steps in endocytosis. Here we show that the yeast endocytic protein Pan1p binds to and activates the Arp2/3 complex. Genetic interactions between PAN1 and mutants of Arp2/3 subunits, or of the Arp2/3 activator LAS17, provide evidence for this activity in vivo. We suggest that Pan1p forms the core of an endocytic complex and physically couples actin polymerization nucleated by the Arp2/3 complex to the endocytic machinery, thus providing the forces necessary for endocytosis.

Clinical presentation and antiviral therapy for poxvirus infection in pudu (Pudu puda).

A severe poxvirus infection occurred in three pudu (Pudu puda), resulting in two fatalities. Cutaneous ulcers with mucopurulent exudate were present around the eyes and nose, at the lip margins, coronary bands, and teats. Mucosal ulcers were present in the oral cavity, esophagus, and forestomachs. In the two fatalities, a secondary disseminated fungal infection also occurred. Affected animals were leukopenic, hypocalcemic, and hyperphosphatemic and had elevated serum alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase levels. Electron microscopic examination of affected skin confirmed the presence of a poxvirus. Neutralizing antibody titers to this virus were present in the two pudu tested. One case was treated with cidofovir, 5 mg/kg i.v. q7d for four treatments. Complete recovery occurred in the treated animal. This is the second report of poxvirus infection in pudu and the first report describing clinical presentation, presence of secondary disseminated fungal infection, and successful treatment.

Identical twins with mental retardation, dysarthria, progressive spastic paraplegia, and brachydactyly type E: a new syndrome or variant of Fitzsimmons-Guilbert syndrome?

We report on concordantly affected female identical twins with mental retardation, dysarthria, progressive spastic paraplegia, and brachydactyly type E. The most similar condition reported is the syndrome described by Fitzsimmons and Guilbert in uniovular twins characterized by progressive spastic paraplegia, dysarthria, brachydactyly type E, and cone-shaped epiphyses. During the last 11 years a report of only one other patient with this syndrome has been published; hence, its phenotypic delineation may be only partial. Although our patients might expand the phenotypic spectrum of this syndrome, they may represent a new disorder.

Familial dystonia and choreoathetosis in three generations associated with bilateral striatal necrosis.

Nine cases of dystonia and choreoathetosis (six females and three males) have developed in three generations of a single family. There has been one death. Neuropathologic examination disclosed bilateral striatal necrosis. In this family, the neurologic disorder has evolved gradually or in association with a febrile illness. There has been no neurologic recovery. The disease is worse in females, has been transmitted only through females, and shows incomplete penetrance and anticipation. The maternal inheritance pattern suggests either an autosomal dominant trait also affecting male reproductive ability or a defect involving the mitochondrial genome.

Periarticular hyperostosis and renal disease in six black lemurs of two family groups.

Proliferative periosteal disease was identified in 6 black lemurs (Eulemur macaco macaco) of 2 family groups. Bilaterally symmetric formation of periosteal new bone at the metaphyseal regions of major long bones was first detected at the stifle and tarsal areas and was detected later at the carpal areas. Bony changes were accompanied by progressive renal disease. The syndrome progressed for 6 to 16 months before the lemurs were euthanatized because of debility. Necropsy revealed changes confined to the skeleton and kidneys. Formation of new bone was detected at all affected joints, and chronic renal disease was evident in each lemur. A specific cause was not identified. Although indistinguishable histologically from hypertrophic osteoarthropathy, several important differences were apparent. Distribution of the periosteal new bone was in the metaphyseal rather than diaphyseal areas. Thoracic or gastrointestinal lesions, typically seen with hypertrophic osteoarthropathy, were not detected, and substantial renal disease was evident. A genetic component may be involved in the development of this condition.

Stage-specific cuticular proteins of Ostertagia circumcincta and Ostertagia ostertagi.

In this study we have shown that NHS-biotin and I125-streptavidin can detect cuticular polypeptides of Ostertagia spp. The labelled polypeptide profile of intact nematodes is simple compared to the profile obtained by labelling homogenates. None of the major internal polypeptides are labelled and the subset of proteins labelled in intact nematodes appears to be mainly surface associated. The results presented here demonstrate that NHS-biotin may be used as a reagent for the analysis of surface polypeptides. The surface polypeptide profiles of the five major developmental stages (L1, L2, L3, L4 and adult) of Ostertagia circumcincta show a series of stage-specific molecules with no polypeptides common to all stages, indicating that the cuticle is a dynamic structure which changes throughout the life cycle. Similarily comparison of Ostertagia ostertagi L3 and L4 stage surface profiles showed that each stage is clearly distinct; comparison of these stages between the two species shows an overall similarity.

Somatosensory evoked potentials predict neuromotor outcome after periventricular hemorrhage.

Thirty-nine very low-birthweight (VLBW) preterm infants with periventricular hemorrhage (PVH) were studied with short-latency median nerve somatosensory evoked potentials (SEP) at two, four and/or six months corrected age, and subsequently were followed to a mean age of 22 months. All 12 infants with a single SEP showing unilateral absence or prolonged latency of the early cerebral (N1) response had motor abnormalities at follow-up. A single normal SEP predicted normal motor development in 19 of 36 infants; two normal SEPs did so in 15 of 26 infants, and three normal SEPs in 12 of 14 infants. These results demonstrate that SEPs play a useful rôle in predicting neuromotor outcome for VLBW preterm infants with PVH.

Angelman's (happy puppet) syndrome: clinical, CT scan and serial electroencephalographic study.

Of four patients having Angelman's syndrome admitted to a state mental facility who were clinically and electroencephalographically evaluated, 2 patients had CT scan studies of the brain. The most impressive and striking features that help in the diagnosis are the mental and physical retardation, nondevelopment of speech despite adequate visual and auditory function, various types of seizures, and episodic uncontrollable laughter. The CT scans of the brain did not offer any clue as to the pathogenesis. The EEGs appeared to fall into two groups: in one an arrest of electrical maturation occurred between ages 1 and 3 and in the other a slow but progressive maturation was evident.

Sydenham's chorea: clinical, EEG, CT scan, and evoked potential studies.

An 8-year-old child developed acute mental confusion, and choreoathetosis without a preceding history of acute rheumatic fever. Serial EEGs showed focal suppression of sleep spindle activity over the right cerebral hemisphere along with high-amplitude polymorphic slow-wave complexes. CT scan studies on 4 occasions and MRI scan failed to show focal lesions corresponding to the clinical manifestations. Results of brainstem auditory evoked potential studies were normal. The short- and long-latency somatosensory evoked potential patterns were abnormal during the acute phase of illness as well as during the follow-up, when the patient was clinically intact. The persistent EEG and evoked potential abnormalities in the presence of clinical recovery from chorea raised questions as to the existence of a subclinical disease process.

Intragenic reversion mutations that improve export of maltose-binding protein in Escherichia coli malE signal sequence mutants.

Escherichia coli strains harboring malE signal sequence point mutations accumulate export-defective precursor maltose-binding protein (MBP) in the cytoplasm. Beginning with these mutants, a number of spontaneous intragenic revertants have been obtained in which export of the MBP to the periplasm is either partially or totally restored. With a single exception, each of the reversion mutations resulted in an increase in the overall hydrophobicity of the signal peptide hydrophobic core by one of five different mechanisms. In some revertants, MBP export was achieved at a rate comparable to the wild type MBP; in other cases, the rate of MBP export was significantly slower than wild type. The results indicate that the overall hydrophobicity of the signal peptide, rather than the absolute length of its uninterrupted hydrophobic core, is a major determinant of MBP export competency. An alteration at residue 19 of the mature MBP also has been identified that provides fairly efficient suppression of the export defect in the adjacent signal peptide, further suggesting that important export information may reside in this region of the precursor protein.

Cerebral cortical isolation in infantile neuroaxonal dystrophy.

Three patients with infantile neuroaxonal dystrophy (INAD, Seitelberger's disease) studied between ages 2 and 5 years, with the characteristic electroencephalographic pattern of high-voltage, fast (16-24 c/sec) rhythms and absence of reactivity on eye-opening or closure (Radermecker and Dumon-Radermecker 1972), also showed no changes in response to intermittent photic stimulation and absence of evoked potentials to flashes, clicks or median nerve stimuli. Although some theta rhythms and delta activity appeared during drowsiness and sleep, the fast rhythms persisted as the dominant feature. There were no central transients or K-complexes. When the patients cried, with hyperventilation, and also during breath-holding spells, slow rhythms appeared and the fast rhythm was reduced. These findings are interpreted as evidence of cerebral cortex isolation, the fast rhythm representing the spontaneous ("idling") activity of the cortex largely disconnected from subcortical or remote cortical influences by the slowly progressive, selective degeneration of axons, characteristic of the pathology of INAD, but the cortex remains responsive to chemical influences. These electrophysiological features become established furing the third year of age, prior EEGs being normal and later ones showing paroxysmal and other abnormal features in addition to the fast rhythm.