Intra-articular injection of mesenchymal stem cells expressing coagulation factor ameliorates hemophilic arthropathy in factor VIII-deficient mice.

BACKGROUND: Transplantation of cells overexpressing a target protein represents a viable gene therapeutic approach for treating hemophilia. Here, we focused on the use of autologous mesenchymal stem cells (MSCs) expressing coagulation factor for the treatment of coagulation factor VIII (FVIII) deficiency in mice. METHODS AND RESULTS: Analysis of luciferase gene constructs driven by different promoters revealed that the plasminogen activator inhibitor-1 (PAI-1) gene promoter coupled with the cytomegalovirus promoter enhancer region was one of the most effective promoters for producing the target protein. MSCs transduced with the simian immunodeficiency virus (SIV) vector containing the FVIII gene driven by the PAI-1 promoter expressed FVIII for several months, and this expression was maintained after multiple mesenchymal lineage differentiation. Although intravenous injection of cell supernatant derived from MSCs transduced with an SIV vector containing the FVIII gene driven by the PAI-1 promoter significantly increased plasma FVIII levels, subcutaneous implantation of the MSCs resulted in a transient and weak increase in plasma FVIII levels in FVIII-deficient mice. Interestingly, intra-articular injection of the transduced MSCs significantly ameliorated the hemarthrosis and hemophilic arthropathy induced by knee joint needle puncture in FVIII-deficient mice. The therapeutic effects of a single intra-articular injection of transduced MSCs to inhibit joint bleeding persisted for at least 8 weeks after administration. CONCLUSIONS: MSCs provide a promising autologous cell source for the production of coagulation factor. Intra-articular injection of MSCs expressing coagulation factor may offer an attractive treatment approach for hemophilic arthropathy.

Induction of factor VIII-specific unresponsiveness by intrathymic factor VIII injection in murine hemophilia A.

SUMMARY BACKGROUND: Hemophilia A is a congenital bleeding disorder caused by a deficiency of coagulation factor VIII. Approximately 30% of hemophilia A patients develop inhibitors against FVIII following replacement therapy. We have reported that neonatal exposure of FVIII antigen can induce antigen-specific immune tolerance by interferon-gamma (IFN-gamma)-dependent T-cell anergy in hemophilia A mice. OBJECTIVE: The thymus plays crucial roles in self-tolerance, with negative selection of self-reactive effector T cells and positive selection of self-reactive regulatory T cells. We investigated the possibility of the induction of antigen-specific immune tolerance by intrathymic injection of FVIII in hemophilia A mice. METHODS: Hemophilia A mice were injected with recombinant FVIII into the thymus under real-time high-resolution image guidance. RESULTS: Anti-FVIII inhibitory antibody titers in mice challenged with intravenous administration of FVIII were significantly lower in mice (n = 22) that had received thymic FVIII injection than in mice (n = 18) without thymic injection (9.4 +/- 2.3 vs. 122.5 +/- 27.6 BU mL(-1), respectively, P = 0.00078). The CD4(+) T cells from thymic-injected mice could not proliferate or produce interleukin (IL)-2, IL-12 and IFN-gamma in response to FVIII. The CD4(+)CD25(+) T cells generated from thymic-treated mice but not from naïve mice efficiently suppressed the in vitro proliferative response of CD4(+) T cells and blocked the in vivo development of anti-FVIII antibodies in the adoptive transfer. CONCLUSION: These data suggest that intrathymic administration of FVIII could result in immune tolerance by induction of FVIII-specific regulatory T cells.

Preclinical evidence of Alzheimer changes in progressive mild cognitive impairment: a qualitative and quantitative SPECT study.

OBJECTIVES: Baseline brain single-photon emission computed tomography (SPECT) can predict mild cognitive impairment (MCI) patients at risk for progressive MCI (PMCI). METHODS: Twenty-eight subjects [12 MCI, 6 with probable Alzheimer's Disease (AD), and 10 normal subjects] underwent baseline brain SPECT and were clinically followed for a mean period of 36 months. RESULTS: Of 12 MCI patients, 6 progressed to PMCI and 6 remained stable. Baseline SPECT identified asymmetric perfusion reduction in the parahippocampus (-5%), lateral parietal (-8%), and posterior cingulate (-11%) cortices--reductions consistent with that of mild AD--in five of the six PMCI patients. Significant perfusion reduction was observed particularly in the frontal cortices of probable AD when compared with PMCI (P < 0.05). CONCLUSION: Baseline SPECT can identify brain perfusion abnormalities among patients with MCI for progression to PMCI. This imaging modality may aid in MCI treatment stratification.

Studies toward the total synthesis of popolohuanone E: enantioselective synthesis of 8-O-methylpopolohuanone E.

[structure: see text]. 8-O-methylpopolohuanone E (2) was synthesized in a highly convergent manner starting from the cis-fused decalin derivative accessible from the (-)-Wieland-Miescher ketone analogue. The synthetic method features a biogenetic-type annulation of the phenolic and quinone segments to regioselectively construct the central tricyclic ring system as the key step.

Inhibition of beta-lactamases by 6,6-bis(hydroxylmethyl)penicillanate.

beta-Lactamases of classes A and C are the two most prevalent resistant determinants to beta-lactam antibiotics among bacterial pathogens. Both these enzymes pursue different mechanisms for their catalytic processes, highlighted by the fact that the hydrolytic water molecule in each approaches the ester of the intermediary acyl-enzyme species from the opposite ends. 6,6-Bis(hydroxylmethyl)penicillanate was designed as an inhibitor that would impair the approach of the hydrolytic water molecule in either of these enzymes upon formation of the acyl-enzyme species. The design, synthesis, and kinetic evaluation of this inhibitor are disclosed herein.

Stereoselective reduction of alpha-bromopenicillanates by tributylphosphine.

[reaction: see text] Diastereoselective reduction of 6-bromo-6-substituted penicillanate esters has been achieved by treatment with tributylphosphine to give 6-substituted penicillanate esters. This reaction would appear to proceed through a phosphonium beta-lactam enolate species, followed by a diastereoselective protonation. This method has the advantage of being simple to carry out and it is mild, gives high diastereoselectivity, and should tolerate a number of functional groups in the substrates. Implications of these observations are discussed.

6-(hydroxyalkyl)penicillanates as probes for mechanisms of beta-lactamases.

6-(Hydroxyalkyl)penicillanates have proven helpful as probes for the mechanisms of beta-lactamases, enzymes of resistance for beta-lactam antibiotics. The present report summarizes the concepts on design, syntheses and use of these molecules in mechanistic studies of beta-lactamases.

Benzodiazepine receptor imaging with iomazenil SPECT in aphasic patients with cerebral infarction.

To investigate the relationship between prognosis of aphasia and neuronal damage in the cerebral cortex, we evaluated the distribution of central-type benzodiazepine receptor (BZR) binding in post-stroke aphasics with [123I]iomazenil and SPECT. We performed iomazenil SPECT in six aphasic patients (aged from 45 to 75 years; all right-handed) with unilateral left cerebral infarction. Three patients showed signs of Broca's aphasia and the other three Wernicke's aphasia. Cerebral blood flow (CBF) imaging was performed with [123I]iodoamphetamine (IMP). The regions of interest (ROIs) on both images were set in the cerebral cortex, cerebellar cortex and language-relevant area in both hemispheres. Three patients were classified in the mild prognosis group and the other three in the moderate prognosis group. The left language-relevant area was more closely concerned with the difference in aphasic symptoms than the right one in both BZR and CBF distribution, but the ipsilateral to the contralateral ratio (I/C ratio) in the language-relevant areas in the BZR distribution was significantly lower in the moderate prognosis group than in the mild prognosis group, although no difference was seen for these values between the two groups in the CBF distribution. These results suggest that BZR imaging, which makes possible an increase in neuronal cell viability in the cerebral cortex, is useful not only for clarifying the aphasic symptoms but also for evaluating the prognosis of aphasia in patients with cerebral infarction.

[Cerebral blood flow of patients with age-associated memory impairment and the early stage of Alzheimer's disease. A study by SPECT using the ARG method].

In order to further understand the pathology of Alzheimer's disease (AD), we have utilized image analysis in diagnosing the early stages of AD in patients with cognitive disorders. CT and MRI, however, have not been feasible since only atrophy is seen and it is difficult to differentiate the changes in AD from age associated changes. In this study we tried to determine whether regional cerebral blood flow (rCBF) measurements using single photon emission CT (SPECT) are feasible for the early diagnosis of AD. Regional CBF (rCBF) was measured using SPECT in three subject groups: Age-associated memory impairment (AAMI. n = 9), mild AD (n = 16), and normal aged patients (mean age = 68.3; n = 20). The subjects were then observed for three years. The region of interest (ROI) for the medial temporal lobe was set at OM-30 degrees to cover the maximum area of the hippocampus. The absolute values of rCBF in the frontal, temporal, and parietal lobes and the cerebellum were significantly lower in the mild AD subjects than in the normal aged subjects. A significant decrease in rCBF was also seen in the medial temporal lobe in both the AD and the AAMI subjects compared to the normal controls. During the three years of follow up, no cases of dementia were seen in the AAMI subjects. However, there were two patients who appeared to have difficulty in adapting to daily life due to amnesia, one with a decrease in rCBF of the medial temporal lobe on the second SPECT, and the other showing a low rCBF the first time. This study suggests that AAMI subjects may comprise both AD and normal subjects. Therefore a more prospective study is needed.

[A quantitative study of regional cerebral blood flow in patients with dementia of Alzheimer type using single photon emission computed tomography--clinical application of the IMP autoradiographic method (IMP ARG method)].

We quantitatively measured regional cerebral blood flow (rCBF) in 37 patients with dementia of Alzheimer type (DAT) to investigate the clinical utilities of the N-isopropyl-p-[123I]iodoamphetamine autoradiographic method (IMP ARG method) that is a quantitative method more simplified and less invasive for IMP-SPECT developed by Iida et al. A given standard input function and a given value of distribution volume (Vd) used for the rCBF measurement of this method were calculated from the dynamic study by six normal volunteers. Mean values [SD] of rCBF (ml/ 100 g/min) in the Cerebral Cortex were 49.0 [6.0] in the controls (n = 20), 42.6 [5.9] in mild DAT group (n = 14), 36.7 [5.5] in moderate DAT group (n = 12), and 26.4 [7.5] in severe DAT group (n = 11), respectively. These values were significantly different between each neighboring group. Moreover, the correlations between the score by the Hasegawa dementia scale (HDS-R) and each rCBF were significant in the temporal, parietal, and frontal cortex. These findings suggest that the rCBF measurement in IMP-SPECT using this method is useful for the diagnosis of the clinical severity in patients with DAT.

Conformation and disulfide bond formation of the constant fragment of an immunoglobulin light chain: effect of truncation at the C-terminal region.

Constant fragments with different carboxyl terminals, CL(109-211), CL(109-207), and CL-(109-200), were prepared by limited carboxypeptidase P or Y proteolysis of the constant fragment, CL-(109-214), of a type lambda immunoglobulin light chain, and their conformations and stabilities, and formation of the disulfide bond from the reduced fragments, were studied. No change in conformation or stability was observed on removal of three residues from the C-terminal end. Removal of seven or more residues from the C-terminal end destabilized the CL fragment. The rate of disulfide bond formation from reduced CL(109-207) was about 7 times faster than that for CL(109-214). These findings suggest that elongation of the polypeptide chain at least beyond the 207th residue is necessary for folding of the CL fragment into a definite conformation.