Cytomegalovirus infection in HIV-infected patients in the era of combination antiretroviral therapy.

BACKGROUND: Cytomegalovirus infection dramatically decreased with the introduction of antiretroviral therapy. Whether incidence, clinical characteristics and prognosis of cytomegalovirus in HIV infected patients, has changed over time is. scarcely known. METHODS: Retrospective single-center study. Patients included in this study were all HIV infected patients that went to our center for any disease, and were diagnosed with cytomegalovirus, during the period 2004-2015. epidemiological, clinical and laboratory patients variables were collected in a clinical database. Clinical characteristics, incidence of cytomegalovirus and predictors of mortality during the study were assessed. Results were considered statistically significant when p < 0.05. All statistical analyses were calculated by SPSS version 20.0 (Chicago, IL,USA). RESULTS: Fifty-six cases of cytomegalovirus infection, in HIV infected patients were identified during the study period (incidence rate-1.7 cases per 1000 persons/year). The most frequent presentation was systemic illness in 43% of cases. Of note,no patients presented with ophthalmic manifestations. The 30-days mortality was 18%. Predictors of mortality were, in the univariate analysis, admission to the intensive care unit OR 32.4 (3.65-287.06) p = 0.0001, and mechanic ventilation 84 OR (8.27-853.12) p = 0.0001, and ART OR 4.1 (0.97-17.31) p = 0.044. These variables were assessed by multivariate analysis, and only mechanical ventilation was statistically significant (p < 0.05) CONCLUSION: Incidence of cytomegalovirus infection was higher than described in the antiretroviral therapy era. Clinical presentation has changed. Mechanic ventilation predicted mortality.

First isolation of West Nile virus in Zambia from mosquitoes.

Mosquito surveillance studies to identify mosquito-borne flaviviruses have identified West Nile Virus (WNV) for the first time in Zambia. The Zambian WNV isolate from Culex quinquefasciatus mosquitoes collected in the Western Province was closely related genetically to WNV lineage 2 South African strains which have been previously shown to be highly neuroinvasive. These data provide the first evidence of the circulation of WNV in Zambia and suggest there should be an increased awareness of possible associated human and animal diseases in that country.

Full blood count values as a predictor of poor outcome of pneumonia among HIV-infected patients.

BACKGROUND: To evaluate the predictive value of analytical markers of full blood count that can be assessed in the emergency department for HIV infected patients, with community-acquired pneumonia (CAP). METHODS: Prospective 3-year study including all HIV-infected patients that went to our emergency department with respiratory clinical infection, more than 24-h earlier they were diagnosed with CAP and required admission. We assessed the different values of the first blood count performed on the patient as follows; total white blood cells (WBC), neutrophils, lymphocytes (LYM), basophils, eosinophils (EOS), red blood cells (RBC), hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, red blood cell distribution width (RDW), platelets (PLT), mean platelet volume, and platelet distribution width (PDW). The primary outcome measure was 30-day mortality and the secondary, admission to an intensive care unit (ICU). The predictive power of the variables was determined by statistical calculation. RESULTS: One hundred sixty HIV-infected patients with pneumonia were identified. The mean age was 42 (11) years, 99 (62%) were male, 79 (49%) had ART. The main route of HIV transmission was through parenteral administration of drugs. Streptococcus pneumonia was the most frequently identified etiologic agent of CAP The univariate analysis showed that the values of PLT (p < 0.009), EOS (p < 0.033), RDW (p < 0.033) and PDW (p < 0.09) were predictor of mortality, but after the logistic regression analysis, no variable was shown as an independent predictor of mortality. On the other hand, higher RDW (OR = 1.2, 95% CI 1.1-1.4, p = 0.013) and a lower number of LYM (OR 2.2, 95% CI 1.1-2.2; p = 0.035) were revealed as independent predictors of admission to ICU. CONCLUSION: Red blood cell distribution and lymphocytes were the most useful predictors of disease severity identifying HIV infected patients with CAP who required ICU admission.

Ectopic expression of c-myc fails to overcome downregulation of telomerase activity induced by herbimycin A, but ectopic hTERT expression overcomes it.

Correction to: Leukemia (2000); 14: 1260­1265; doi: 10.1038/sj.leu.2401828. Since the publication of the above article the authors have identified an error in Figure 1. Figure 1 shows the modulation of telomerase activity by herbimycin A in K562 cells: (a) cell cycle and (b) telomerase activity, mRNA expressions of hTERT, hTERC, TEP-1, c-myc, cyclin D1 and b-actin, and c-Myc protein. The authors however wish to inform the readers that Figure 1b incorrectly shows hTERT mRNA, which is the result of herbimycin A treatment of cyclin-D1-transfected K562 cells (Figure 3b, hTERT mRNA). While preparing Figure 1, the authors mistakenly submitted a figure that used the incorrect photo data following confusion regarding file names. The correct figure can be found below: The authors wish to apologise for any inconvenience caused and confirm that the conclusions drawn from this research are not affected by this error.

Oral administration of recombinant human acid α-glucosidase reduces specific antibody formation against enzyme in mouse.

Animal and human studies of enzyme replacement therapy for Pompe disease have indicated that antibodies generated against the infused recombinant human acid α-glucosidase (rhGAA) can negatively impact therapeutic outcome. In this study, we show that oral administration of rhGAA into mice can reduce the titer of anti-rhGAA antibody following immunization with rhGAA. Oral administration of rhGAA is safe and antigen specific, it offers advantages over other immunosuppressive drugs.

Neonatal gene transfer using lentiviral vector for murine Pompe disease: long-term expression and glycogen reduction.

Pompe disease results from the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA), leading to accumulated glycogen in the heart and the skeletal muscles, which causes cardiomyopathy and muscle weakness. In this study, we tested the feasibility of gene therapy for Pompe disease using a lentivirus vector (LV). Newborn GAA knockout mice were treated with intravenous injection of LV encoding human GAA (hGAA) through the facial superficial temporal vein. The transgene expression in the tissues was analyzed up to 24 weeks after treatment. Our results showed that the recombinant LV was efficient not only in increasing the GAA activity in tissues but also in decreasing their glycogen content. The examination of histological sections showed clearence of the glycogen storage in skeletal and cardiac muscles 16 and 24 weeks after a single vector injection. Levels of expressed hGAA could be detected in serum of treated animals until 24 weeks. No significant immune reaction to transgene was detected in most treated animals. Therefore, we show that LV-mediated delivery system was effective in correcting the biochemical abnormalities and that this gene transfer system might be suitable for further studies on delivering GAA to Pompe disease mouse models.

Systemic administration of a PEGylated adenovirus vector with a cancer-specific promoter is effective in a mouse model of metastasis.

Cancer gene therapy by adenovirus vectors (Advs) for metastatic cancer is limited because systemic administration of Adv produces low therapeutic effect and severe side effects. In this study, we generated a dual cancer-specific targeting vector system by using PEGylation and the telomere reverse transcriptase (TERT) promoter and attempted to treat experimental metastases through systemic administration of the vectors. We first optimized the molecular size of PEG and modification ratios used to create PEG-Ads. Systemic administration of PEG-Ad with 20-kDa PEG at a 45% modification ratio (PEG[20K/45%]-Ad) resulted in higher tumor-selective transgene expression than unmodified Adv. Next, we examined the effectiveness against metastases and side effects of a TERT promoter-driven PEG[20K/45%]-Ad containing the herpes simplex virus thymidine kinase (HSVtk) gene (PEG-Ad-TERT/HSVtk). Systemic administration of PEG-Ad-TERT/HSVtk showed superior antitumor effects against metastases with negligible side effects. A cytomegalovirus (CMV) promoter-driven PEG[20K/45%]-Ad also produced antimetastatic effects, but these were accompanied by side effects. Combining PEG-Ad-TERT/HSVtk with etoposide or 5-fluorouracil enhanced the therapeutic effects with negligible side effects. These results suggest that modification with 20-kDa PEG at a 45% modification ratio is the optimal condition for PEGylation of Adv, and PEG-Ad-TERT/HSVtk is a prototype Adv for systemic cancer gene therapy against metastases.

Clinical manifestations and natural history of Japanese heterozygous females with Fabry disease.

Fabry disease is an X linked lysosomal storage disorder resulting from deficiency of α-galactosidase A activity. Although disease manifestations in heterozygous females with Fabry disease have been considered to be rare and mild, heterozygous patients with severe symptoms have recently been reported. In this study we focused on the clinical characteristics of Japanese females patients with Fabry disease and analysed data from 36 women. Data on clinical manifestations such as acroparaesthesia, hypohidrosis, angiokeratoma, corneal opacities, proteinuria, end-stage renal disease (ESRD), left ventricular hypertrophy (LVH) and cerebral infarction were obtained by questionnaire; these analysed by the Kaplan-Meier method and compared with the data from Japanese male patients. Eighty-six per cent of female patients had at least one clinical sign of Fabry disease. Their most common symptoms were acroparaesthesia and corneal opacities, and diagnosis was most commonly based on family history. The cumulative incidences of angiokeratoma, corneal opacities, proteinuria, ESRD, LVH and cerebral infarction were significantly lower in female than in male patients. However, corneal opacities, proteinuria and LVH developed progressively in female patients as well as in male patients. The incidence of LVH was especially high in older female patients and the cumulative incidence of LVH in women over 68 years of age was 100%. We consider that the term X-linked 'recessive' is inappropriate for Fabry disease because of the high prevalence of its pathognomonic signs in heterozygous female patients. Careful and long-term evaluation of clinical signs is important in heterozygous females patients with Fabry disease.

Identification of Fabry's disease by the screening of alpha-galactosidase A activity in male and female hemodialysis patients.

BACKGROUND: Although previous studies reported that the prevalence of Fabry's disease was 0.16 - 1.2% in hemodialysis (HD) patients based on measurement of a-galactosidase A (alpha-Gal A) activity, few reports detected female patients by the screening for alpha-Gal A. Here we determined the prevalence of Fabry's disease not only in male but also in female HD patients by measuring alpha-Gal A. METHODS: Plasma alpha-Gal A was measured in 696 consecutive males (n = 401) and females (n = 295) on HD. Patients with low plasma alpha-Gal A were examined for leukocyte alpha-Gal A, and patients with low leukocyte alpha-Gal A underwent alpha-Gal A gene sequence analysis for possible mutations, and family survey. RESULTS: Among 15 patients with low plasma alpha-Gal A activity, 4 male patients with low leukocyte alpha-Gal A and 1 female patient revealing low plasma alpha-Gal A were detected in 696 HD patients (0.7% of total patients). 3 of these 5 patients were already diagnosed to have the classical type of Fabry's disease. The other 2 patients were newly diagnosed as Fabry's disease, and did not have typical manifestations of Fabry's disease other than renal failure and left ventricular hypertrophy. DNA analysis of these 2 newly diagnosed patients revealed that each had an alpha-Gal missense mutation, previously identified (E66Q, M2961). CONCLUSION: Fabry's disease should be considered in the etiology of unexplained end-stage renal disease. Not only affected males but also affected females undergoing HD patients can be readily diagnosed by alpha-Gal A activities and gene analysis. These patients and their family members may benefit from enzyme replacement therapy for Fabry's disease.

Enzyme replacement therapy in Japanese Fabry disease patients: the results of a phase 2 bridging study.

Fabry Disease (alpha-galactosidase A deficiency) is an X-linked hereditary disorder leading to the pathological accumulation of globotriaosylceramide (GL-3) in lysosomes, particularly in the vascular endothelium of the kidney, heart and brain. We report the results of an open-label phase 2 study that was undertaken to evaluate whether ethnic differences exist that would affect agalsidase beta (Fabrazyme) treatment of Fabry patients in the Japanese population, relative to safety and efficacy. The study design mirrored the design of the completed phase 3 clinical trial that led to approval of the product agalsidase beta. The 13 Japanese, male Fabry patients enrolled in the study received the enzyme replacement therapy over a period of 20 weeks as biweekly infusions. All selected efficacy end points showed improvements that were comparable with findings from the phase 3 study. These improvements included reductions of GL-3 accumulation in both kidney and skin capillary endothelial cells to (near) normal levels (92% of patients). Kidney and plasma GL-3 levels decreased by 51.9% and 100%, respectively, by ELISA. Renal function remained normal. Fabry-associated pain, and quality of life, showed improvement over baseline in multiple categories. Related adverse events were mild or moderate in intensity and mostly infusion-associated (fever and rigors). As expected, IgG antibody formation was observed in 85% of the patients, but had no effect on treatment response. These results suggest that treatment with agalsidase beta is safe and effective in Japanese patients with Fabry disease. With regard to safety and efficacy, no differences were observed as compared to the caucasian population.

Brain transplantation of genetically modified bone marrow stromal cells corrects CNS pathology and cognitive function in MPS VII mice.

Current therapies for lysosomal storage diseases (LSDs), enzyme replacement therapy and bone marrow transplantation are effective for visceral organ pathology of LSD, but their effectiveness for brain involvement in LSDs is still a subject of controversy. As an alternative approach, we transplanted genetically modified bone marrow stromal (BMS) cells to lateral ventricle of newborn mucopolysaccharidosis VII (MPS VII) mice. MPS VII is one of LSDs and caused by deficiency of beta-glucuronidase (GUSB), resulting in accumulation of glycosaminoglycans (GAGs) in brain. At 2 weeks after transplantation, the GUSB enzyme-positive cells were identified in olfactory bulb, striatum and cerebral cortex, and the enzymatic activities in various brain areas increased. The GAGs contents in brain were reduced to near normal level at 4 weeks after transplantation. Although GUSB activity declined to homozygous level after 8 weeks, the reduction of GAGs persisted for 16 weeks. Microscopic examination indicated that the lysosomal distention was not found in treated animal brain. Cognitive function in MPS VII animals as evaluated by Morris Water Maze test in treated mice showed a marked improvement over nontreated animals. Brain transplantation of genetically modified BMS cells appears to be a promising approach to treat diffuse CNS involvement of LSDs.

High gene expression of the mutant adenovirus vector, both in vitro and in vivo, with the insertion of integrin-targeting peptide into the fiber.

In the present study, a first-generation adenovirus (Ad) vector was modified with the RGD peptide inserted into the fiber. The insertion of an integrin-targeting sequence into the Ad vector notably enhanced the luciferase expression in the Coxsackie virus and Adenovirus Receptor-deficient A2058 and B16BL6 melanoma cells. The results of an in vivo study with tumor-bearing mice also showed that Ad-RGD-Luc had enhanced gene expression in many organs and in the B16BL6 tumor compared to that induced by a conventional Ad vector after intravenous injection.

Treatment of lysosomal storage disorders: cell therapy and gene therapy.

Most lysosomal storage diseases have central nervous system (CNS) involvement. No effective treatment is available at present. We investigated the usefulness of brain-directed gene therapy and cell therapy using mouse models of lysosomal storage diseases. For gene therapy to the CNS, a recombinant adenovirus encoding beta-galactocerebrosidase gene was injected into the cerebral ventricle of neonatal twitcher mice, a murine model of Krabbe disease. Improvements in neurological symptoms and a prolonged lifespan were observed. Brain activity of beta-galactocerebrosidase was increased significantly and the concentration of a cytotoxic metabolite, psychosine, was decreased. Pathological observations of the brain were also improved in treated twitcher mice. For cell therapy to the CNS, a neural stem cell line derived from human fetal brain was genetically engineered to overexpress beta-glucuronidase and transplanted into the cerebral ventricles of neonatal MPS VII mice, a model of beta-glucuronidase deficiency. Transplanted human neural stem cells were found to integrate and migrate in the host brain and to produce large amounts of beta-glucuronidase. Brain contents of the substrate of beta-glucuronidase were reduced and widespread clearing of lysosomal storage was observed in treated MPS VII mice. These data suggest that brain-directed gene/cell therapy may be useful in the treatment of neurological alterations in lysosomal storage diseases.

Tumor-suppressive activities by chemokines introduced into OV-HM cells using fiber-mutant adenovirus vectors.

In this study, fiber-mutant adenovirus vectors encoding chemokines, Ad-RGD-mCCL17, Ad-RGD-mCCL21 and Ad-RGD-mCCL22 were constructed. The insertion of integrin-targeting RGD sequence into fiber knob of adenovirus vectors notably enhanced the infection efficiency into tumor cells. Among three chemokine-encoding vectors evaluated, Ad-RGD-mCCL22 showed significant tumor-suppressive activity via transduction into OV-HM cells.

Adenovirus-mediated overexpression of a gene prevents hearing loss and progressive inner hair cell loss after transient cochlear ischemia in gerbils.

The use of adenoviral vectors has recently provided a novel strategy for direct gene transfer into the cochlea. In this study, we assessed the utility of an adenoviral vector expressing glial-cell-derived neurotrophic factor (GDNF) in ischemia-reperfusion injury of the gerbil cochlea. The vector was injected through the round window 4 days before ischemic insult. The distribution of a reporter transgene was confirmed throughout the cochlea from the basal to the apical turn and Western blot analysis indicated significant upregulation of GDNF protein 11 days following virus inoculation. Hearing ability was assessed by sequentially recording compound action potentials (CAP), and the degree of hair cell loss in the organ of Corti was evaluated in specimens stained with rhodamine-phalloidin and Hoechst 33342. On the seventh day of ischemia, the CAP threshold shift and inner hair cell loss were remarkably suppressed in the Ad-GDNF group compared with the control group. These results suggest that adenovirus-mediated overexpression of GDNF is useful for protection against hair cell damage, which otherwise eventually occurs after transient ischemia of the cochlea.

Adenovirus-mediated prenatal gene transfer to murine central nervous system.

In some lysosomal storage disorders pathological alterations in the central nervous system (CNS) occur as early as the prenatal period and the neuropathology progresses rapidly soon after birth. In these diseases, postnatal therapies alone are often insufficient. Therefore prenatal gene therapy to the CNS may be necessary. In order to investigate the feasibility of gene transfer to the CNS prenatally, we administered recombinant adenovirus carrying LacZ gene to rat embryos from embryonic day 9 to 12 (E9-E12). Results showed that efficient transduction of the reporter gene to the CNS was achieved when adenoviruses were injected at E12. The regions where the reporter gene was transduced mainly localized at the telencephalon and hypophysis of the embryo, and the gene expression persisted at least 1 week after birth. In addition, when adenoviruses were injected at E9, E10 and E11, no transgene expression was detected in the CNS, but was mainly observed in the liver, the heart and the skin, respectively.

Activin A increases the bone mass of grafted bone in C3H/HeJ mice.

Activin A, a member of the TGF-b superfamily, is abundant in bone matrix, but little is known about its physiological role in bone metabolism. The present study was undertaken to determine whether topical activin A can increase the bone mass of isografted bone. The tibiae were bilaterally dissected from a donor C3H/HeJ mouse and transplanted subcutaneously in the dorsal region of a recipient mouse. One isografted tibia was topically infused for either 1, 2, 3, or 4 weeks with activin A, using an osmotic minipump at a dose of 0.02, 0.2, or 2 ng/hr. The other tibia was infused with 0.9% NaCl (control). The following results were obtained: (1) Topical activin A (2 ng/hr) stimulated periosteal bone formation after 2 or 3 weeks. The bone area in a standardized transverse section averaged 1.3 fold that in the control. (2) Numerous cuboidal or conical osteoblasts appeared on the surface of newly formed bone after the infusion of activin A for 2 or 3 weeks. Autoradiographic studies using 3H-proline revealed that the surface area of newly formed bone labelled with autoradiographic silver grains was greater in activin A-treated bone than in the control, suggesting an increased synthesis and secretion of collagen by osteoblasts. (3) Topical activin A increased the number of osteoclasts after 2 to 4 weeks. Furthermore, enhanced or increased bone resorption was observed in the projected anterior site of activin A-treated bone after 4 weeks. These results suggest that topical activin A increases the bone mass of isografted bone by increasing bone turnover.