Oncogene-independent resistance in Philadelphia chromosome - positive (Ph+) acute lymphoblastic leukemia (ALL) is mediated by activation of AKT/mTOR pathway.

Tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, dasatinib, and ponatinib have significantly improved the life expectancy of Philadelphia chromosome-positive (Ph+) acute lymphocytic leukemia (ALL) patients; however, resistance to TKIs remains a major clinical challenge. Point mutations in the tyrosine kinase domain (TKD) of BCR-ABL1 have emerged as the predominant cause of acquired resistance. In approximately 30% of patients, the mechanism of resistance to TKIs remains elusive. This study aimed to investigate mechanisms of nonmutational resistance in Ph+ ALL. Here we report the development of a nonmutational resistance cell line SupB15-RT; conferring resistance to approved ABL kinase inhibitors (AKIs) and allosteric inhibitors GNF-2, ABL001, and crizotinib, except for dasatinib (IC90 50nM), a multitarget kinase inhibitor. We found that the AKT/mTOR pathway is activated in these cells and their proliferation inhibited by Torin-1 with an IC50 of 24.7 nM. These observations were confirmed using 3 different ALL patient-derived long term cultures (PDLTCs): (1) HP (BCR-ABL1 negative), (2) PH (BCR-ABL1 positive and responsive to TKIs) and (3) BV (BCR-ABL1 positive and nonmutational resistant to TKIs). Furthermore, Torin-1 and NVP-BEZ235 induced apoptosis in PH and BV cells but not in HP cells. Our experiments provide evidence of the involvement of AKT/mTOR pathway in the evolution of nonmutational resistance in Ph+ ALL which will assist in developing novel targeted therapy for Ph+ ALL patients with BCR-ABL1 independent nonmutational resistance.

Isolation by culture and PCR identification of LipL32 gene of pathogenic Leptospira spp. in wild rats of Kuala Lumpur.

BACKGROUND: A study was conducted to confirm the status of rats as the carrier of pathogenic leptospira in Kuala Lumpur, Malaysia. METHOD: A total of 140 urine samples were collected from trapped rats. These samples were cultured in EMJH enriched media and 18 of these samples (12.9%) were found to be positive when observed under x40 by dark field microscope. Genomic DNA was extracted from all the 18 native isolates for PCR. RESULT: All the 18 isolates generated the expected 786 base pair band when the set of primers known to amplify LipL32 gene were utilized. These results showed that the primers were suitable to be used for the identification of pathogenic leptospira from the 18 rat samples. CONCLUSION: The sequencing of the PCR products and BLAST analysis performed on each representative isolates confirmed the pathogenic status of all these native isolates as the LipL32 gene was detected in all the Leptospira isolates. This indicates that the rats are carriers of the pathogenic leptospira in the study area, and therefore are of public health importance.

Sociodemographic factors associated with IgG and IgM seroprevalence for human cytomegalovirus infection in adult populations of Pakistan: a seroprevalence survey.

BACKGROUND: The seroprevalence of human cytomegalovirus (HCMV) infection ranges from 30 to 90 % in developed countries. Reliable estimates of HCMV seroprevalence are not available for Pakistan. This study determined the seroprevalence and sociodemographic factors associated with HCMV infection in adult populations of Karachi, Pakistan. METHODS: A seroprevalence survey was conducted on 1000 adults, including residents of two semi-urban communities, and visitors to a government and a private hospital. Questionnaire-based interviews were conducted. Sera were analysed for HCMV-specific IgG and IgM. Chi-square or Fisher's exact test was used for comparing sociodemographic variables against seropositivity of HCMV-IgG or IgM. Multiple logistic regression modeling was performed for IgG seroprevalence and adjusted odds ratios were computed. RESULTS: The seroprevalence of HCMV-IgG and IgM was 93.2 and 4.3 % respectively. 95.3 % of individuals who were IgM seropositive were also seropositive for IgG. Around 6 % (15/250) of women of childbearing age remained uninfected and were therefore susceptible to primary infection. HCMV-IgG seroprevalence was associated with being female (p = 0.001), increasing age (p = 0.002) and crowding index (p = 0.003) and also with lower levels of both education (p < 0.001) and income (p = 0.008). Seroprevalence also differed significantly by marital status (p = 0.008) and sampling location (p < 0.001). A logistic regression model for HCMV-IgG seroprevalence showed associations with being female (OR = 1.89; 95 % CI: 1.10-3.25), increasing age (OR = 3.95; 95 % CI: 1.79-8.71) and decreasing income (OR = 0.72; 95 % CI: 0.54-0.96). A strong association was observed between increased seroprevalence of HCMV-IgM and decreasing household size (p = 0.008). CONCLUSIONS: Seroprevalence of HCMV is very high in Pakistan, although 6 % of women of childbearing age remain at risk of primary infection. The IgM seropositivity observed in some individuals living in small household size (1-3 individuals) with persistent HCMV infection could have resulted from a recurrent HCMV infection. Future longitudinal research in pregnant women and neonates is required to study the trends in HCMV seroprevalence over time in Pakistan for the development of a potential HCMV prevention and vaccination programme.

Benchmarking the Self-Assembly of Surfactin Biosurfactant at the Liquid-Air Interface to those of Synthetic Surfactants.

The adsorption of surfactin, a lipopeptide biosurfactant, at the liquid-air interface has been investigated in this work. The maximum adsorption density and the nature and the extent of lateral interaction between the adsorbed surfactin molecules at the interface were estimated from surface tension data using the Frumkin model. The quantitative information obtained using the Frumkin model was also compared to those obtained using the Gibbs equation and the Langmuir-Szyszkowski model. Error analysis showed a better agreement between the experimental and the calculated values using the Frumkin model relative to the other two models. The adsorption of surfactin at the liquid-air interface was also compared to those of synthetic anionic, sodium dodecylbenzenesulphonate (SDBS), and nonionic, octaethylene glycol monotetradecyl ether (C14E8), surfactants. It has been estimated that the area occupied by a surfactin molecule at the interface is about 3- and 2.5-fold higher than those occupied by SDBS and C14E8 molecules, respectively. The interaction between the adsorbed molecules of the anionic biosurfactant (surfactin) was estimated to be attractive, unlike the mild repulsive interaction between the adsorbed SDBS molecules.

Self-assembly of a surfactin nanolayer at solid-liquid and air-liquid interfaces.

Surfactin, a sustainable and environmentally friendly surface active agent, is used as a model to study the adsorption of biosurfactants at hydrophobic and hydrophilic solid-liquid interfaces as well as the air-liquid interface. Surfactin adsorption was monitored as a function of time and concentration using surface plasmon resonance (SPR) technique in the case of the solid-liquid interfaces or the drop shape analysis (DSA) technique in the case of the air-liquid interface. The results obtained in this study showed that surfactin adsorption at the "hard" hydrophobic (functionalized with octadecanethiol) solid-liquid and the "soft" air-liquid interface were 1.12 ± 0.01 mg m(-2) (area per molecule of 157 ± 2 Å(2)) and 1.11 ± 0.05 mg m(-2) (area per molecule of 159 ± 7 Å(2)), respectively, demonstrating the negligible effect of the interface "hardness" on surfactin adsorption. The adsorption of surfactin at the hydrophilic (functionalized with ß-mercaptoethanol) solid-liquid interface was about threefold lower than its adsorption at the hydrophobic-liquid interfaces, revealing the importance of hydrophobic interaction in surfactin adsorption process. The affinity constant of surfactin for the investigated interfaces follows the following order: air > octadecanethiol > ß-mercaptoethanol. Biosurfactants, such as surfactin, are expected to replace the conventional fossil-based surfactants in several applications, and therefore the current study is a contribution towards the fundamental understanding of biosurfactant behavior, on a molecular level, at hydrophobic and hydrophilic solid-liquid interfaces in addition to the air-liquid interface. Such understanding might aid further optimization of the utilization of surfactin in a number of industrial applications such as enhanced oil recovery, bioremediation, and detergency.

Clinicopathological correlation of primary and recurrent choroidal neovascularisation following surgical excision in age related macular degeneration.

AIMS/BACKGROUND: Fluorescein angiography and histopathological findings were correlated in two patients with recurrent choroidal neovascular membranes (CNVs) in an attempt to gain insight into the possible causes of recurrent CNVs and into the healing response after CNV excision. METHODS: Two patients with recurrent CNVs underwent repeat excision, and the excised tissue was studied with light and electron microscopy. RESULTS: Incomplete CNV excision probably led to the recurrences. The portion initially excised appears to have been anterior to the RPE in case 1. In both cases, recurrent CNVs contained RPE-like like cells suggesting that native RPE can repopulate the dissection bed. The tissue excised at the second operation contained areas with hyperplastic RPE and fragments of Bruch's membrane (external to the RPE basement membrane) in a matrix of fibrillar collagen and fibrocytes, suggesting that initial removal of the CNV can be followed by an abnormal anatomical arrangement of RPE and scarring of Bruch's membrane. CONCLUSIONS: Abnormal resurfacing of the dissection bed by RPE and fibroblasts may underlie, in part, the limited visual outcome often seen after surgical excision of CNVs in age related macular degeneration.

Decreased choriocapillaris perfusion following surgical excision of choroidal neovascular membranes in age-related macular degeneration.

AIMS/BACKGROUND: To evaluate macular changes following surgical excision of subfoveal choroidal neovascular membranes (CNVs) in age-related macular degeneration (AMD). METHODS: The clinical records, fluorescein angiograms, and CNV histopathology of 12 patients with AMD who underwent surgical excision of subfoveal CNV were reviewed. RESULTS: New areas of decreased choriocapillaris perfusion were noted by fluorescein angiography in the previous location of the CNV in 8/12 (75%) cases. Surgically excised tissue contained retinal pigment epithelium (RPE) in 11/11 specimens and choriocapillaris in 1/11 specimen studied. CONCLUSIONS: Choriocapillaris atrophy may partly underlie the limited visual outcome following subfoveal surgery for AMD. Abnormal choriocapillaris perfusion following CNV excision may be due to pre-existing choriocapillaris atrophy, to choriocapillaris damage or removal at the time of surgery, or to RPE removal at surgery with abnormal RPE repopulation of the dissected area and subsequent choriocapillaris degeneration.

Recombinant hirudin prevents postoperative fibrin formation after experimental cataract surgery.

PURPOSE: The authors studied the effect of a direct acting antithrombin agent, desulfatohirudin variant 1 (Revasc, Ciba-Geigy, Ltd., Basel, Switzerland), on postoperative fibrin formation after cataract surgery in rabbits. METHODS: Phacoemulsification was performed in a masked fashion on 28 eyes of 28 New Zealand white rabbits. Ten control group eyes had lactated Ringer's solution in the infusion and an intracameral injection (approximately 1.5 ml) at the end of the case. Ten group 1 eyes received hirudin 100 micrograms/ml in the infusion and intracameral injection. Eight group 2 eyes had 100 micrograms/ml hirudin in the intracameral injection only. Using slit-lamp examination, all eyes were graded for the amount of fibrin and intraocular hemorrhage in a masked fashion on postoperative day 1. RESULTS: Comparison of the mean postoperative fibrin grade (0-4) in group 1 (mean = 0.3), group 2 (mean = 0.25) and the control group (mean = 3.4) revealed a statistically significant difference between hirudin-treated and control eyes (P = 0.0002 for group 1, P = 0.0005 for group 2). No intraocular hemorrhage was noted in any group. CONCLUSIONS: Recombinant hirudin significantly decreases postoperative fibrin formation in a rabbit cataract extraction model. Intracameral injection of hirudin alone appears to be at least as effective as infusion of hirudin throughout the case. With further study, this agent has potential for use in cataract surgery on patients known to be at high risk for postoperative fibrin formation.

Intraocular injection of recombinant hirudin to prevent experimental postoperative fibrin.

PURPOSE: To test the efficacy of a single intraocular injection of various doses of recombinant desulphatohirudin variant 1 (Revasc) in a rabbit model after lensectomy-vitrectomy surgery. METHODS: Standard fragmatome lensectomies and core vitrectomies were performed on Dutch Belted rabbits. In a masked fashion, 17 control eyes received single intraocular injections of lactated Ringer's solution, and the treated eyes, with eight eyes per treatment group, received single intraocular injections of hirudin at doses of 0.02 microgram, 2 micrograms, 10 micrograms, 50 micrograms, or 200 micrograms. RESULTS: Intraocular injections of hirudin were effective in preventing postoperative fibrin formation at doses of 2 micrograms. There was no notable intraoperative bleeding. With an injection of 2.0 micrograms hirudin there was no hemorrhage at any time after surgery; with an injection of 200 micrograms hirudin, however, there was notable bleeding. CONCLUSION: Hirudin is effective in the prevention of postoperative fibrin formation with a single intraocular injection of 2.0 micrograms after lensectomy-vitrectomy surgery in the rabbit model. There was no bleeding at the effective dose of 2.0 micrograms; at 100 times the effective dose, however, there was significant postoperative bleeding.

Recombinant hirudin for prevention of experimental postoperative intraocular fibrin.

PURPOSE: To determine the efficacy of a specific antithrombin agent (recombinant desulphatohirudin variant 1 [Revasc, Ciba-Geigy, Ltd., Basel, Switzerland]) administered in the infusion fluid to prevent early postoperative fibrin formation in a rabbit lensectomy and vitrectomy model. METHODS: Standard fragmatome lensectomies and core vitrectomies were performed prospectively in a masked fashion on ten control eyes with lactated Ringer's infusion and on ten eyes treated with 10 microgram of recombinant hirudin/ml in the infusate. The amounts of fibrin and hemorrhage were graded in a masked fashion by using slit-lamp examination and indirect ophthalmoscopy on postoperative days 1 through 5 and on day 7. RESULTS: The difference in the mean grade of fibrin formed on the first postoperative day in the eyes treated with recombinant hirudin (mean, 0.9) in relation to the mean grade of fibrin in the control eyes (mean, 3.5) was statistically significant (P = .004). This difference was also significant on the second postoperative day (P = .01). None of the treated eyes developed intraoperative or postoperative hemorrhage. CONCLUSIONS: Recombinant desulphatohirudin variant 1 is an effective inhibitor of postoperative fibrin formation in a rabbit model and is not associated with an increased risk of intraoperative or postoperative bleeding at the tested dose. This drug may be a useful adjunct in vitreous surgery for both proliferative vitreoretinopathy and the complications of proliferative diabetic retinopathy.

Cytomegalovirus retinitis associated with Hodgkin's disease.

BACKGROUND: Patients with intraocular involvement by Hodgkin's disease may present with a variety of ocular abnormalities. Intraocular infections also have been reported in these patients, including two cases of cytomegalovirus retinitis (CMV) diagnosed on postmortem examination. It often is difficult to differentiate between these conditions on clinical examination. METHODS: A patient with Hodgkin's disease presented with an atypical retinitis, vasculitis, and a serous macular detachment. Results of clinical examination, fluorescein angiography, and vitreous biopsy for culture and polymerase chain reaction (PCR) testing are described. RESULTS: The diagnosis of an atypical CMV retinitis simulating possible intraocular involvement by Hodgkin's disease was confirmed by PCR testing of undiluted vitreous. Intravenous therapy with foscarnet, guided by the results of the vitreous biopsy, resulted in resolution of the retinitis and an improvement in vision. CONCLUSION: Intraocular involvement by cytomegalovirus retinitis can simulate intraocular lymphoma in a patient with Hodgkin's disease Polymerase chain reaction testing was useful in establishing the diagnosis and guiding successful therapy in this patient.

Multiple endocrine neoplasia type III.

The multiple endocrine neoplasia (MEN) syndromes are autosomally dominant inherited disorders in which hyperplastic or neoplastic changes occur in a wide variety of tissues. The specific syndromes are classified according to the endocrine glands affected. MEN type I consists of an aggregation of tumors of parathyroid, pancreatic, and pituitary glands. The association of medullary carcinoma of the thyroid (MCT) and pheochromocytoma is called MEN type II or type IIA, and if combined with mucosal neuromas, intestinal ganglioneuromatosis, and prominent corneal nerves, is named MEN type III or type IIB. Individuals afflicted with MEN type III are characterized by a marfanoid habitus, mucosal neuromas involving oral and ocular tissues, and a number of ophthalmologic findings including prominent corneal nerves, thickened eyelids, and subconjunctival neuromas. These features are easily recognized during the ocular exam, allowing the ophthalmologist to make an early diagnosis of this syndrome prior to the onset of life-threatening manifestations like medullary thyroid carcinoma and pheochromocytoma.