Resting CD4(+) T cells with CD38(+)CD62L(+) produce interleukin-4 which contributes to enhanced replication of T-tropic human immunodeficiency virus type 1

A significant increase in the CD38(+) population among T lymphocytes has been observed in human immunodeficiency virus type 1 (HIV-1)-infected carriers. We previously reported a higher replication rate of T-tropic HIV-1 in the CD4(+)CD38(+)CD62L(+) than CD38(-) subset under conditions of mitogen stimulation after infection. Here, we revealed a similarly high susceptibility in the CD38(+) subset on culture with conditioned medium containing Th2 cytokine, interleukin (IL)-4 that was produced endogenously from this subset on stimulation with mitogen or anti-CD3 antibody for 3 days. The contribution of IL-4 to the upregulated production of virus in the CD38(+) subset was confirmed by culture of this subset with recombinant human IL-4. In contrast, the rate of replication in the CD38(-) subset was not augmented in the conditioned medium from either subset or with IL-4. However, there were no differences in the surface expression of IL-4 receptor or HIV-1 receptors CD4 and CXCR4 between the two subsets. Thus, the CD4(+)CD38(+)CD62L(+) subset comprises a specific cell population secreting endogenous Th2 cytokine that contributes to the efficient production of T-tropic HIV-1 through upregulation at a certain stage of the viral life cycle, probably after the adsorption step.     

Prognostic factors for successful surgical outcome with preoperative prism adaptation test in patients with superior oblique palsy.

PURPOSE: This study evaluated the surgical outcome of patients managed with preoperative prism adaptation test (PAT) and investigated prognostic factors for successful motor alignment in adult patients with superior oblique palsy. METHODS: Prospective study of preoperative PAT was performed. Fifty-seven patients with superior oblique palsy, aged 16 to 81 years, participated in this study. Patients were assigned to surgery with the target angle based on either the original angle or the prism compensated angle. When the amount of neutralizing prism exceeded 4delta or more compared to the original angle of deviation, the patient was defined as having prism compensation, and the target angle for surgery was based on the amount of neutralizing prism. The motor success rate was compared between the 2 groups at the 3-month postoperative follow-up. RESULTS: The prism responders group showed a superior outcome compared to that of the prism non-responders group (77% successful outcome compared with 46%, p = 0.0397). The presence of prism compensation and the amount of vertical deviation were significant prognostic factors for successful motor alignment. CONCLUSION: Preoperative PAT is a useful prognostic indicator of successful surgical outcome in patients with superior oblique palsy.     

PDGFRA mutations in gastrointestinal stromal tumors: frequency, spectrum and in vitro sensitivity to imatinib.

PURPOSE: Gastrointestinal stromal tumors (GISTs) commonly harbor oncogenic mutations of the KIT tyrosine kinase, which is a target for the kinase inhibitor imatinib. A subset of GISTs, however, contains mutations in the homologous kinase platelet derived growth factor receptor alpha (PDGFRA), and the most common of these mutations is resistant to imatinib in vitro. Little is known of the other types of PDGFRA mutations that occur in GISTs. MATERIALS AND METHODS: We determined the KIT and PDGFRA mutation status of 1,105 unique GISTs using a combination of denaturing high-performance liquid chromatography and direct sequencing. RESULTS: 66 in exon 18, 11 in exon 12, and three in exon 14. Transient expression of representative PDGFRA isoforms in CHO cells revealed imatinib sensitivity of exon 12 mutations (SPDHE566-571R and insertion ER561-562) and an exon 14 substitution (N659K). However, most isoforms with a substitution involving codon D842 in exon 18 (D842V, RD841-842KI, DI842-843IM) were resistant to the drug, with the exception of D842Y. Interestingly, other mutations in exon 18 (D846Y, N848K, Y849K and HDSN845-848P) were all imatinib sensitive. Proliferation studies with BA/F3 cell lines stably expressing selected PDGFRA mutant isoforms supported these findings. CONCLUSION: Including our cases, there are 289 reported PDGFRA-mutant GISTs, of which 181 (62.6%) had the imatinib-resistant substitution D842V. However, our findings suggest that more than one third of GISTs with PDGFRA mutations may respond to imatinib and that mutation screening may be helpful in the management of these tumors.     

Effect of Switching Therapy to Pegaptanib in Eyes With the Persistent Cases of Exudative Age-Related Macular Degeneration

Purpose of this study was to evaluate the efficacy of switching to pegaptanib monotherapy for persistent cases of exudative age-related macular degeneration (AMD).Out of 296 eyes of 296 patients treated with ranibizumab or ranibizumab combined with photodynamic therapy (PDT), 50 eyes of 50 AMD patients were found to be resistant to these treatments. Over a 12-month period, intravitreal pegaptanib (IVP) 0.3 mg was administered at intervals of 6 weeks until the exudation disappeared prospectively. All patients were examined with the following tests: best-corrected visual acuity (BCVA) and central retinal thickness (CRT), determined at the initial visit, before the first IVP (baseline), and at 12 months. The factors responsible for achieving dry macula with IVP were examined statistically.The rate of persistent cases with intravitreal ranibizumab (IVR) and/or PDT was 17.0%. The mean number of IVPs administered was 5.4 (range, 2–9). Logarithm of the minimal angle of resolution BCVA at 12 months was stable or improved by ≥0.3 in 49 eyes (98.0%), with a significant improvement noted between the baseline and final BCVA (P = 0.01, paired t test). The CRT (mean ± standard deviation) was 446.9 ± 150.6 µm at the initial visit, 414.5 ± 146.5 µm at baseline, and 318.7 ± 99.0 µm at 12 months. There was a significant decrease in the mean CRT between the measurements at baseline and at 12 months after the first IVP (P = 0.002, Bonferroni correction). At 12 months, the exudative change was completely resolved in 27 eyes (54.0%) and reduced in 21 eyes (42.0%). The number of previous IVR treatments was significantly correlated with dry macula at 12 months.After switching therapy to pegaptanib in persistent cases of AMD, most patients maintained or improved their BCVA and exhibited a positive treatment response at 12 months.     

Subretinal injection of recombinant tissue plasminogen activator for submacular hemorrhage associated with ruptured retinal arterial macroaneurysm

Purpose

To evaluate the surgical outcomes of small-gauge vitrectomy with subretinal injection of recombinant tissue plasminogen activator (rt-PA) for a submacular hemorrhage caused by a ruptured retinal arterial macroaneurysm (RAM).

Methods

Non-comparative, consecutive case-series performed at two ophthalmological institutions. We examined 22 eyes of 22 patients with a submacular hemorrhage associated with a RAM but without a preretinal or sub-internal limiting membrane hemorrhage at the fovea. During 25-gauge vitrectomy, approximately 4000–8000 IU of rt-PA was injected subretinally, followed by the injection of air or 10 % sulfur hexafluoride as a tamponade. The patients maintained an upright position for 1 hour, then turned to a facedown position for 1 to 3 days. The best-corrected visual acuity (BCVA) and postoperative complications were evaluated.

Results

The average interval from the onset of symptoms to surgery was 8.4?±?7.6 days, and the average size of the subretinal hemorrhage was 3.4?±?1.0 disc diameters. The submacular hemorrhage was displaced from the foveal area in all eyes after 1 week. The mean baseline BCVA was 1.41?±?0.41 logMAR units, and it improved to 0.91?±?0.43 at 1 month and to 0.64?±?0.45 at the final visit (P?=?0.0001, P?<?0.0001 respectively). A macular hole was detected intraoperatively in two eyes and postoperatively in two eyes, and both were closed by internal limiting membrane peeling or a second vitrectomy.

Conclusions

Small-gauge vitrectomy with subretinal rt-PA injection and gas tamponade were effective in displacing a submacular hemorrhage associated with a RAM.

     

Genetic analysis of two female patients with incomplete Denys-Drash syndrome

Denys-Drash syndrome (DDS) is characterized by genital anomaly, early onset nephropathy and high risk for developing Wilms' tumor (WT). Recently, mutations in exon 8 or 9 of the Wilms' tumor suppressor gene (WT1) have been found in the majority of DDS patients studied. We analyzed these two exons of the WT1 gene in genomic DNA from two female patients with DDS by using polymerase-chain reaction (PCR) and direct sequencing. The patients were accompanied with normal external genitalia, early onset renal failure between 6 and 12 months of age, and unilateral Wilms' tumor. Genomic DNA was isolated from peripheral blood leucocytes of the patients. Amplification of exons 8 and 9 of the WT1 gene by PCR was performed, and direct sequencing of the PCR product was performed using an automatic DNA sequencer. Two heterozygous missense mutations were found in these patients, including a missense mutation in exon 9 at codon 388 replacing the wild-type Cys with Phe, and a previously described mutation in exon 9 at codon 398 replacing the wild-type Leu with Pro. Cys388Phe is a novel mutation in the WT1 gene in the DDS. These cases are considered to be "incomplete DDS" with nephropathy and Wilms' tumor and without genital anomaly, the validity of which has been confirmed by mutation analysis.     

Inhibition of calcium oxalate crystal growth in vitro by uropontin: another member of the aspartic acid-rich protein superfamily.

The majority of human urinary stones are primarily composed of calcium salts. Although normal urine is frequently supersaturated with respect to calcium oxalate, most humans do not form stones. Inhibitors are among the multiple factors that may influence the complex process of urinary stone formation. We have isolated an inhibitor of calcium oxalate crystal growth from human urine by monoclonal antibody immunoaffinity chromatography. The N-terminal amino acid sequence and acidic amino acid content of this aspartic acid-rich protein, uropontin, are similar to those of other pontin proteins from bone, plasma, breast milk, and cells. The inhibitory effect of uropontin on calcium oxalate crystal growth in vitro supports the concept that pontins may have a regulatory role. This function would be analogous to that of other members of the aspartic acid-rich protein superfamily, which stereospecifically regulate the mineralization fronts of calcium-containing crystals.     

A mutation in the extracellular cysteine-rich repeat region of the beta3 subunit activates integrins alphaIIbbeta3 and alphaVbeta3

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, alphaIIbbeta3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of alphaIIbbeta3. One clone (AM-1) expressed constitutively active alphaIIbbeta3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antialphaIIbbeta3 antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of beta3(T562N) was responsible for receptor activation. In fact, T562N also activated alphaVbeta3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated alphaIIbbeta3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of beta3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to alphaIIbbeta3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125(FAK), indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of beta3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.     

Evaluation of leukocyte dynamics in choroidal circulation with indocyanine green-stained leukocytes

PURPOSE: To develop a new method with which to visualize leukocytes moving through the choroidal vessels of pigmented animals and enable the evaluation of leukocyte dynamics in the choroidal microcirculation. METHODS: Pigmented rabbits and monkeys were used in this study. Leukocytes, collected by centrifugal separation of autologous blood, were stained with indocyanine green (ICG) dye. The ICG-stained leukocyte fluid was injected into the vein, and the fundus image was obtained with a scanning laser ophthalmoscope. The image was recorded on videotapes and analyzed with a personal computer-based image analysis system. RESULTS: In pigmented rabbits, fluorescent leukocytes moving in the choroidal circulation were clearly visible for more than 1 hour. In monkeys, distinct fluorescent dots were seen moving approximately 50 to 200 microm in the foveal avascular zone for more than 30 minutes after the injection of the ICG-stained leukocyte fluid. Dim fluorescent dots were seen moving in the fundus. Although the movement of these dim dots was difficult to trace, they seemed to be moving in the choroidal vessels. In the rabbits, the mean flow velocity of leukocytes moving without plugging was 0.48 +/- 0.14 mm/sec in the peripheral choriocapillaris. In the monkeys, the mean flow velocity of distinct fluorescent leukocytes without plugging was 2.45 +/- 0. 48 mm/sec in the posterior choroid. CONCLUSIONS: In pigmented rabbits and monkeys, this method allows visualization of leukocytes passing through the choroidal vessels and provides a new way to investigate, noninvasively and in vivo, leukocyte dynamics in the choroidal microcirculation.     

Large Bielschowsky head-tilt phenomenon and inconspicuous vertical deviation in the diagnostic positions in congenital superior oblique palsy

PURPOSE: To report a case of congenital superior oblique palsy with an unusually large Bielschowsky head-tilt phenomenon (BHP) and disproportional inconspicuous vertical deviation. METHODS: Case report. RESULTS: An 18-year-old woman presented with slight compensatory head tilting and a Bielschowsky head-tilt phenomenon of 50 Delta on left tilting. Magnetic resonance imaging revealed atrophy of the left superior oblique muscle. A Hess screen test showed a slight underaction of the left superior oblique muscle, but neither an obvious overaction of the ipsilateral inferior oblique muscle nor inhibitory palsy of the contralateral superior rectus muscle was found. With a 3-mm recession of the ipsilateral superior rectus muscle, Bielschowsky head-tilt phenomenon decreased to 25 Delta. CONCLUSION: A large Bielschowsky head-tilt phenomenon was possibly caused by an increased gain of the otolith-ocular reflex affecting the vertical rectus muscle.