August 22, 2010

Hepatitis carriers will get right to be teachers

An HBV carrier shows his health certificate in this file photo taken in 2009. Photo: CFP

Source: Global Times [09:04 August 20 2010]
By Yang Jie

Hepatitis virus carriers in Beijing will be able to work as kindergarten teachers in the future, as authorities have cancelled restrictions against them.

The Beijing Municipal Health Bureau and Beijing Municipal Commission of Education jointly issued a notice on Wednesday expunging the rule that hepatitis virus carriers of all types were not suitable to be engaged in preschool education. In 2001, that limitation was added to the physical examination standards for teachers' qualification confirmations.

"[The notice] means hepatitis virus carriers will be treated equally," said Zhao Guowei, a publicity department officer from the education commission. "They were not allowed to teach kindergarten children before."

This move comes after a previous notice was jointly released by three ministries to preserve hepatitis B virus (HBV) carriers' schooling and working rights in January. It stated that HBV tests should be cancelled and educational organizations and employers are prohibited from carrying out HBV tests, or even inquiring about people's HBV statuses.

The Beijing No.1 Kindergarten said that in the past, applicants diagnosed as hepatitis virus carriers would not be employed. "All staff members have to receive an annual physical examination, and those not meeting the examination standards would be asked to receive treatment until they recovered," director Feng Huiyan told the Legal Mirror.

Feng said they have not received an official notice regarding revisions to the teachers' physical examination requirements, and Zhao said he is also unclear of when the new regulation would come into effect.

A 31-year-old woman surnamed Zhou, whose 4-year-old daughter is in grade 2 in a Beijing State-owned kindergarten, said she welcomed this new policy. "Maybe some parents will show their concerns toward children's safety. It's understandable," she said. "The most important thing is that authorities should further popularize hepatitis knowledge."

According to the official notice released in January, HBV can only be transmitted through blood, sex and maternal-neonatal transmission, and activities of daily life and work would not lead to virus transmission.

Source

War on drugs: Bring out the peace pipe

Editorial
The Guardian, Wednesday 18 August 2010
Article history

Sir Ian Gilmore is a distinguished physician. Nicholas Green is a leading barrister. Pillars of society, they share a radical opinion: they believe drugs should be decriminalised – not from any dogmatic position but from their own experience in medicine and the law.

Sir Ian, a liver specialist and the outgoing president of the Royal College of Physicians, told the BBC yesterday that current policy aggravated the harms associated with drug abuse and cited approvingly a BMJ article by Stephen Rolles of the pro-legalisation organisation Transform. In June Mr Green suggested that if the government was serious about cutting the prison population it should consider decriminalising individual drug use.

When the UN first sounded the alarm about the global drugs trade in 1961, it warned of the threat it posed to the world's health. It was President Nixon, swiftly backed in Britain, who converted the concern into a moral crusade. His war on drugs, both nationally and internationally, has caused harm that far exceeds the unquestionable damage of drug abuse. In ripples and surges from Mexico's catastrophic turf wars to gangland murders in Detroit and drive-by shootings in Birmingham, civil society is undermined and in places destroyed by the profitable lawlessness of the illegal drugs trade. It is time to sue for peace.

This is a global war, and ultimately it needs a global solution. The first step has to be to acknowledge that the moral evil is drug trafficking, not drug abuse. The best way to undermine the traffickers is to tackle demand for their product. And as part of holistic policy that has to tackle wellbeing more widely, decriminalising individual drug use would be a good start. Portugal, where drug use was decriminalised nearly 10 years ago, is showing the way. Its evidence suggests the most persuasive argument against changing policy – that it would increase the numbers abusing drugs – is baseless. There has been no significant increase in drug use, while take-up of treatments has increased and health has improved.

Politicians could prepare public opinion for change by a public assessment of what Britain's war on drugs has achieved. It should ask whether better results could have come by a less damaging route. A policy that results, via the Afghanistan poppy harvest, in financial support for the Taliban, boosts international organised crime and is the underlying problem for more than half of the UK prison population will require some defending. Decriminalisation would not be an answer in itself. Legalisation is no quick fix. But prohibition's defenders need to show how, against its dire results, their policy can still be justified.

Source
Hepatology
Early View (Articles online in advance of print)
Article first published online: 5 AUG 2010
DOI: 10.1002/hep.23796

Darmendra Ramcharran 1,*,§, Abdus S. Wahed 2, Hari S. Conjeevaram 3, Rhobert W. Evans 1,Tianyi Wang 4, Steven H. Belle 1,2, Leland J. Yee 1,5

1 Departments of Epidemiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA
2 Biostatistics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA
3 Division of Gastroenterology, School of Medicine, University of Michigan, Ann Arbor, MI
4 Infectious Disease and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA
5 Division of Infectious Diseases, School of Medicine, University of Pittsburgh, Pittsburgh, PA

The Virahep-C study was conducted as a cooperative agreement funded by the National Institute of Diabetes and Digestive and Kidney Diseases with cofunding from the National Center on Minority Health and Health Disparities and a Cooperative Research and Development Agreement with Roche Laboratories. The specific grant numbers and membership of the Virahep-C Study Group are provided in the primary publication of the study (reference 4). Funding for additional lipid profile analyses using stored serum samples was obtained for an ancillary study of the pathogenesis of steatosis and insulin resistance in chronic hepatitis C virus infection (KL2 RR024154-02 to L. J. Y. from the National Institutes of Health).
 
Email: Darmendra Ramcharran (dramcharran@gmail.com)
*Correspondence: Darmendra Ramcharran, 24 Doylston Drive, Cranston, RI 02905

Potential conflict of interest: Nothing to report.
§ D.R. is currently affilitated with Via Research, LLC, Princeton Junction, NJ

Abstract

Approximately one half of patients who undergo antiviral therapy for chronic hepatitis C virus (HCV) genotype 1 infection do not respond to treatment. African Americans (AAs) are less responsive to treatment than Caucasian Americans (CAs), but the reasons for this disparity are largely unknown. Recent studies suggest that serum lipids may be associated with treatment response. The aims of this study were to evaluate baseline and changes in serum lipids during therapy, determine whether serum lipids are associated with virological response, and assess whether these measures explain the racial difference in efficacy. The study participants were from Virahep-C, a prospective study of treatment-naïve patients with genotype 1 HCV infection who received peginterferon (PEG-IN) alfa-2a plus ribavirin therapy for up to 48 weeks. Fasting serum lipids were analyzed at baseline and during and after therapy in 160 AAs and 170 CAs. A relative risk (RR) model was employed to evaluate characteristics associated with sustained virological response (SVR). Antiviral therapy was associated with changes in serum lipids during and after antiviral therapy, with the changes differing by race and the amount of PEG-IFN taken. Baseline lipid measures independently associated with higher rates of SVR were lower triglyceride and higher low-density lipoprotein cholesterol, with an interaction between high-density lipoprotein cholesterol (HDLc) and gender. Lipid measures did not contribute significantly to an explanation of the racial difference in SVR. Conclusion: Serum lipids are associated with SVR, although these paramaters did not explain the racial difference in treatment response. The results of this study are compatible with proposed biological mechanisms of HCV entry, replication, and secretion, and may underscore new potential therapeutic targets for HCV eradication. (Hepatology 2010)

Source

Pharmacotherapy for hepatic encephalopathy.

Drugs. 2010 Jun 18;70(9):1131-48. doi: 10.2165/10898630-000000000-00000.

Phongsamran PV, Kim JW, Cupo Abbott J, Rosenblatt A.

Department of Clinical Pharmacy, University of Southern California School of Pharmacy, USC University Hospital Department of Pharmacy, Los Angeles, California, USA. phongsam@usc.edu

Abstract

Hepatic encephalopathy (HE) is a challenging clinical complication of liver dysfunction with a wide spectrum of neuropsychiatric abnormalities that range from mild disturbances in cognitive function and consciousness to coma and death. The pathogenesis of HE in cirrhosis is complex and multifactorial, but a key role is thought to be played by circulating gut-derived toxins of the nitrogenous compounds, most notably ammonia. Therapeutic treatment options for HE are currently limited and have appreciable risks and benefits associated with their use. Management of HE primarily involves avoidance of precipitating factors, limitation of dietary protein intake, and administration of various ammonia-lowering therapies such as non-absorbable disaccharides and select antimicrobial agents. Non-absorbable disaccharides, such as lactulose, have traditionally been regarded as first-line pharmacotherapy for patients with HE. However, multiple adverse events have been associated with their use. In addition, recent literature has questioned the true efficacy of the disaccharides for this indication. Neomycin, metronidazole and vancomycin may be used as alternative treatments for patients intolerant or unresponsive to non-absorbable disaccharides. Antimicrobials reduce bacterial production of ammonia and other bacteria-derived toxins through suppression of intestinal flora. Neomycin has been reported to be as effective as lactulose, and similar efficacy has been reported with vancomycin and metronidazole for the management of HE. However, the adverse effects frequently associated with these antimicrobials limit their use as first-line pharmacological agents. Neomycin is the most commonly used antimicrobial for HE and, although poorly absorbed, systemic exposure to the drug in sufficient amounts causes hearing loss and renal toxicity. Long-term neomycin therapy requires annual auditory testing and continuous monitoring of renal function. Long-term use of metronidazole has been associated with neurotoxicity in patients with cirrhosis, including dose-dependent peripheral neuropathy. Vancomycin may be a safer option for HE in patients with chronic liver disease; however, limited experience, possible bacterial overgrowth and risk for enteric bacteria resistance preclude the routine use of vancomycin for HE. Rifaximin is a novel antimicrobial agent with a wide spectrum of activity that has shown promise as an alternative antimicrobial treatment option for HE. Several clinical trials have compared rifaximin to the disaccharides, lactulose and lactitol, and the antimicrobial neomycin. Rifaximin appears to be at least as effective as conventional drug therapy and has been associated with fewer adverse effects due to its limited systemic absorption. The available clinical data appear to support a favourable benefit-risk ratio for rifaximin, which has shown efficacy with an improved tolerability profile. Future studies are needed in order to truly characterize its cost effectiveness in today's healthcare environment. Other less frequently utilized alternative treatment options include administration of benzodiazepine receptor antagonists, branched-chain amino acids, ornithine aspartate, zinc supplementation, sodium benzoate, dopamine receptor agonists, acarbose and probiotics. Presently, there is relatively limited clinical data supporting their routine use in HE.

PMID: 20518580 [PubMed - indexed for MEDLINE]

Source

Top 10 States with Highest Cases of Hepatitis for 2009

August 21, 2010 02:51 AM EDT

Known as the inflammation of the liver, hepatitis is classified into three groups namely hepatitis A, hepatitis B and hepatitis C. It is usually manifested by the existence of inflammatory cells specifically in the liver. There are instances when hepatitis can be acute or chronic as well. It can show symptoms common to all viral infections including loss of appetite and dark urine among others.

In the US, there are a total of 2,587 cases reported for 2009. This is broken down into 817 cases of hepatitis A, 1,449 cases of hepatitis B and 321 cases of hepatitis C. The top 10 states affected by the disorder are as follows:

1. California. Reporting a total of 237 cases, California represents 9.16% of the total population inflicted with the disorder. There are 116 cases for hepatitis A, 105 cases for hepatitis B and 16 cases of hepatitis C.

2. Florida. This state reported 236 cases, 1 point short from California's 237. This represents 9.12% of the nation's total and is categorized into 84 cases for hepatitis A, 136 cases for hepatitis B and 16 cases for hepatitis C.

3. Texas. With 217 cases, Texas' hepatitis cases is 8.39% of the country's total. There are 66 cases of hepatitis A in the state while there are 139 cases of hepatitis B. The remaining 12 cases belong to hepatitis C.

4. New York. The state has 128 cases for New York Upstate and New York City. This is 4.94% of the total cases for the country. This is divided into 48 cases for hepatitis A, 61 cases for hepatitis B and 19 cases for hepatitis C.

5. Connecticut. With 112 cases, Connecticut has 4.33% hepatitis incidents out of the country's total. This is broken down into 43, 26 and 23 cases for hepatitis A, B, and C respectively.

6. North Carolina. Reporting 104 cases for this disease (31, 56 and 17 cases each for hepatitis A, B and C), the state represents 4.02% of the country's overall numbers.

7. Michigan. The state has a total of 103 cases representing 32, 59 and 12 for hepatitis A, B and C respectively. This is 3.98% of the total for the entire country.

8. New Jersey. The state has 97 cases which represent 34 for hepatitis A, 57 for hepatitis B and 6 for hepatitis C. This is all in all 3.75% of US's total for 2009.

9. Pennsylvania. Tying up with New Jersey, Pennsylvania's cases are broken down as follows: 29 cases for hepatitis A; 52 cases for hepatitis B and 16 cases for hepatitis C.

10. Illinois. Lagging just one case behind New Jersey and Pennsylvania, Illinois has 96 cases for the disease. It reported 46 cases for hepatitis A and another 47 cases for the B-type. As for hepatitis C, the state has 3 cases. This is 3.71% of the country's total.

The total number of hepatitis cases may be way below the total number of people living in the US. While this is so, there is no reason not to take necessary precaution when it comes to this illness.

Lena Butler, the author of Health & Drug Testing Information Center a longer version of this article is located at Top 10 States with Highest Cases of Hepatitis for 2009, and resources from other home health and wellness testing articles are used such as Hepatitis C Test.

Source
The Journal of Infectious Diseases 2010;202:000–000
© 2010 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2010/20207-00XX$15.00
DOI: 10.1086/656212

MAJOR ARTICLE

Elijah Paintsil, 1,2 Huijie He, 3 Christopher Peters, 4 Brett D. Lindenbach, 4 and Robert Heimer 3

Departments of 1 Pediatrics, 2 Pharmacology, 3 Epidemiology and Public Health, and 4 Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut

Background.We hypothesized that the high prevalence of hepatitis C virus (HCV) among injection drug users might be due to prolonged virus survival in contaminated syringes.

Methods.We developed a microculture assay to examine the viability of HCV. Syringes were loaded with blood spiked with HCV reporter virus (Jc1/GLuc2A) to simulate 2 scenarios of residual volumes: low void volume (2 μL) for 1‐mL insulin syringes and high void volume (32 μL) for 1‐mL tuberculin syringes. Syringes were stored at 4°C, 22°C, and 37°C for up to 63 days before testing for HCV infectivity by using luciferase activity.

Results.The virus decay rate was biphasic ( h and h). Insulin syringes failed to yield viable HCV beyond day 1 at all storage temperatures except 4°, in which 5% of syringes yielded viable virus on day 7. Tuberculin syringes yielded viable virus from 96%, 71%, and 52% of syringes after storage at 4°, 22°, and 37° for 7 days, respectively, and yielded viable virus up to day 63.

Conclusions.The high prevalence of HCV among injection drug users may be partly due to the resilience of the virus and the syringe type. Our findings may be used to guide prevention strategies.

Received 7 January 2010; accepted 14 April 2010; electronically published 20 August 2010.

(See the editorial commentary by Rich and Taylor, on pages XXX–XXX.)

Reprints or correspondence: Dr Elijah Paintsil, Departments of Pediatrics and Pharmacology, Yale University School of Medicine, 333 Cedar St, New Haven, CT 06520 (elijah.paintsil@yale.edu).

The global burden of morbidity and mortality from hepatitis C virus (HCV) infection is truly pandemic [1]. There is no vaccine for the prevention of HCV infection, and current therapeutic regimens for HCV infection are limited by efficacy, cost, and treatment adverse effects. Therefore, reduction of risk associated with HCV transmission remains the primary strategy for curbing the HCV epidemic. HCV is transmitted primarily through percutaneous exposure to blood contaminated with HCV. The prevalence of HCV is disproportionately high among injection drug users (IDUs), with seroprevalence as high as 95% [2–11]. The transmission of HCV and human immunodeficiency virus (HIV) among IDUs has been associated with the sharing of equipment used to prepare and administer drugs [12–14]. The prevalence of HCV among IDUs exceeds that of HIV across all seroprevalence studies in many countries. Even in locations in the United States where HIV seroprevalence among IDUs is low (1%–10%), HCV seroprevalence among IDUs is high (30%–85%) [15–18]

HCV incident infections continue to occur at a startling high rate in IDU populations worldwide. It is estimated that the probability of transmission of HCV per exposure to a contaminated syringe is 5‐fold to 20‐fold higher than that of HIV transmission [19–23]. Although harm reduction programs have effectively reduced the incidence of HIV among IDUs, such reductions in incidence have rarely been observed for HCV [8, 24–26]. The difference in transmission between HCV and HIV may be attributed to a higher infectivity of HCV compared with HIV. The biology of HCV transmission, however, has not been well characterized because of the lack of an efficient cell culture and small animal model for assessing HCV replication and infectivity. To date, polymerase chain reaction (PCR)–based assay for detecting viral RNA has been used as a surrogate for infectivity; there is no direct correlation between nucleic acid concentration and viable virus [27].

We hypothesized that the efficient transmission of HCV among IDUs may be partly due to the ability of the virus to remain viable in contaminated syringes for prolonged periods. To test this hypothesis, we developed a microculture assay that allowed us to propagate HCV from small residual volumes contained in the dead space of syringes used by IDUs, and to determine the effects of storage at different temperatures for prolonged periods on the viability of HCV in syringes. We report the results of the first study, to our knowledge, that simulates HCV transmission among IDUs by directly assaying HCV infectivity in syringes.

MATERIALS AND METHODS

Virus and cells.The construction of the Jc1/GLuc2A reporter virus was similar to that of J6/JFH(p7‐Rluc2A) [28] and has been reported elsewhere [29]. Jc1/GLuc2A is a derivative of the chimeric genotype 2a FL‐J6/JFH [30, 31], with a luciferase gene from Gaussia princeps inserted between the p7 and NS2 genes. Viral stocks of Jc1/GLuc2A reporter virus were prepared by RNA transfection of Huh‐7.5 cells. Four days after transfection, the viral culture media was harvested, clarified by centrifugation at 2000g for 10 min, filtered through 0.2‐μm pore size filters, and stored at −80°C until use. The titer of HCV was quantified by infecting cells with serial dilutions of the stock virus and determining the dilution that will infect 50% (median tissue culture infective dose [TCID50]) of the wells by using the method of Reed and Muench [32].

Highly permissive human heptoma cells (Huh‐7.5 subline) [33] were maintained as subconfluent, adherent monolayers in Dulbecco modified Eagle medium, supplemented with 10% heat‐inactivated fetal calf serum and 1 mmol/L nonessential amino acids (Invitrogen) at 37°C and 5% CO2.

Establishing a standard curve.To test the range of the assay sensitivity, we introduced serial dilutions of HCV into a culture system that included virus and target cells in 96‐well plates. The day before the experiment, 96‐well plates were seeded with Huh‐7 cells/well in 100 μL of medium and incubated at 37°C in 5% CO2. On the day of the experiment, HCV stocks used for the experiment were mixed with ethylenediaminetetraacetic acid (EDTA)‐anticoagualted blood from an HIV and HCV seronegative donor at a ratio of 1:10. Serial 1:2 dilutions of the HCV were made in triplicate. The medium from the wells was gently aspirated from the cells and replaced with 100 μL of the HCV‐blood mixture. After 4 h of incubation, the cells were washed with sterile phosphate‐buffered saline to remove the input virus, and fresh medium was added and incubated for 3 days. After 3 days, culture supernatant was harvested and mixed with 20 μL of lysis buffer before luciferase activity was measured using luciferase assay reagent kit (Promega) and a luminometer (FARCyte; Amersham Biosciences). The relative luciferase activity was determined as a function of HCV infectivity and plotted against concentration of HCV (TCID50/mL) on a logarithmic scale. The set of experiments was performed on 2 separate occasions, and the data were combined for analysis.

HCV decay assay.To investigate the rate of decay of the infectivity of the stock Jc1/Glu2 virus, several 100 μL aliquots of the virus were stored for 0–96 h at room temperature. Aliquots were removed every 6 h or less and stored at −80°C. The stored aliquots were thawed and used to infect the Huh‐7.5 cells. The relative infectivity was determined by measuring the luciferase activity after 3 days of infection as described above.

Syringes.The types of syringes used by IDUs vary. The residual volume that remains in a syringe after injection depends on the size and design of the syringe [34]. For syringes with fixed needles, the void volume (ie, dead‐space fluid) remains in the tip of the syringe, at the base of the plunger, and in the needle itself when the plunger is fully depressed; these syringes generally have a low void volume. Syringes with detachable needles retain fluid in the hub of the syringe, as well as at the base of the plunger and in the needle; these syringes generally have a high void volume. We used 2 different kinds of syringes in our experiments: the low void volume U‐100 1‐mL insulin syringe with an attached 27‐gauge, 0.5‐inch needle and the high void volume 1‐mL tuberculin syringe, with a detachable 26‐gauge, 0.5‐inch needle. The average void volumes after complete depression of the plunger for insulin and tuberculin syringes have been reported elsewhere as 2 and 32 μL, respectively [35, 36]

Viability of HCV in stored syringes.Syringes were loaded with HCV‐spiked blood to replicate the practice of “booting” by IDUs [37, 38]. The usual intravenous injection sequence includes properly registering the syringe in the vein, in which visible blood is drawn into the syringe, then injecting the drug, which leaves a void volume of drug solution mixed with some blood. However, many injectors pull up on the plunger a second time, introducing blood into the barrel of the syringe that mixes with the remaining drug solution. This “booted” material is reinjected, leaving within the syringe a void volume that is predominantly blood. In our experiments, we chose to simulate the practice of booting because it constituted the worst‐case scenario that maximizes the amount of HCV‐contaminated blood.

After preparing syringes to replicate booting, they were either immediately tested for viable virus or stored at 4°C, 22°C, and 37°C for up to 63 days before testing. To test syringes, they were flushed with 100 μL of culture medium, which was introduced into Huh‐7.5 cells in a 96‐well plate and incubated for 3 days. After 3 days, culture supernatant was analyzed for relative luciferase activity as described above.

The range of different storage temperatures has been used in previous HIV studies, and they reflect different ambient temperatures in which injection practices may occur [36]. The storage duration captured the time used syringes remain in circulation in the absence of a syringe exchange program as was measured in New Haven, Connecticut (mean duration, 23.5 days) [26, 39].

Data analysis.The regression analysis for the standard curve comparing infectivity and relative luciferase activity and the exponential decay analysis were determined using GraphPad Prism program (version 4.0; GraphPad Software Inc). The relative luciferase activity and TCID50 were plotted on a logarithmic scale; time of storage was plotted on a linear scale. For determining the decay rate, the slope was used to calculate values.

RESULTS

HCV microculture assay.We developed a microculture assay for investigating the viability of HCV recovered from contaminated syringes. The HCV used (Jc1/Gluc2A) had a luciferase gene from G. princeps inserted between the p7 and NS2 gene [29]. After infection of Huh‐7.5 cells with Jc1/GLuc2A, the GLuc2A enzyme and infectious reporter virus are secreted into the culture medium. HCV replication could be determined over time by measuring secreted GLuc2A activity [29]. This system allowed us to use relative luciferase activity as a function of infectivity or viability of HCV recovered from syringes. Huh‐7.5 cells were infected with HCV reporter virus and incubated for 3 days before culture supernatants were analyzed for luciferase activity.

Assay sensitivity.As a first step, we determined the linear dynamic range of the microculture system. Starting with a viral stock of known TCID50, we prepared serial 1:2 dilutions that were introduced into our system. Negative controls (using HCV‐contaminated blood without cells, uncontaminated blood, or Huh‐7.5 cell alone) yielded uniformly negative results. When 100 μL aliquots of dilution series were introduced, supernatants had relative luciferase activity that showed a strict correlation to expected TCID50 over a linear range of 4 logs, between 1 and 104 TCID50 (Figure 1). This range of sensitivity allowed us to detect changes in infectivity comparable to a >100‐fold reduction in viral load. The limit of detection in the microculture assay was 250 relative luciferase units, equivalent to 0.1 TCID50.


Figure1. Linear dynamic range of microculture assay. Aliquots (100 μL) of serial 1:2 dilutions of stock virus were used to infect Huh 7.5 cells. After 3 days of incubation, the culture supernatant was harvested and the concentration of virus determined as a function of relative luciferase activity. The relative luciferase units (RLU) were plotted as a function of the concentration of input virus (median tissue culture infective dose [TCID50]/mL) on a logarithmic scale. The experiment was performed on 2 separate occasions in triplicate, and the data were combined for analysis.

Decay of infectivity of HCV at room temperature.We next investigated the rate of decay of the infectivity of the stock virus at room temperature. Aliquots of the virus were left in room temperature for up to 96 h. Samples were collected at intervals of 6 h and stored at −80°C until the determination of infectivity. We observed a biphasic decay of HCV viability (Figure 2). There was a rapid decline of infectivity within the first 6 h, with a of 0.4 h, followed by a second phase of a relatively slow exponential decay with a of 28 h.


Figure2. Hepatitis C virus decay rate at room temperature. Aliquots (100 μL) of the virus were stored 0–96 h at room temperature. Aliquots were removed from room temperature every 6 h or less and stored at −80°C. The stored aliquots were thawed and used to infect Huh‐7.5 cells. The relative infectivity was determined by measuring the relative luciferase units (RLU) after 3 days of infection. Each value is the mean of 2 independent experiments.

Survival of HCV in syringes.We last investigated the survival of HCV recovered from syringes stored at different temperatures. We simulated 2 scenarios of residual volumes after complete depression of the plunger: low void volume (2 μL) with 1‐mL insulin syringe (with permanently attached needle) and high void volume (32 μL) with 1‐mL tuberculin syringe (with detachable needle). The syringes were loaded with HCV‐contaminated blood and stored at different temperatures for up to 63 days. For each experiment to test for HCV survival, the contents of at least 15 stored syringes for each combination of storage time and temperature were introduced into our assay system. The proportion of HCV‐positive syringes and the infectivity per HCV‐positive syringe were determined. The results presented here came from at least 3 independent experiments.

The low void volume insulin syringes were stored for up to 14 days. We observed an inverse relationship between temperature and HCV survival (Figure 3A). Both the number of HCV‐positive syringes and infectivity of HCV in the positive syringes declined rapidly over time. We recovered viable HCV from syringes stored at 4° for up to 7 days (5% HCV syringes), whereas syringes stored at 22° and 37° yielded no HCV‐positive syringes beyond day 1 of storage. The loss in infectivity of HCV per positive syringe recovered after 1 day of storage at 22° and 37° was 30% and 96%, respectively. After storage at 4°, HCV‐positive syringes showed 38% and 92% reductions in infectivity after 3 and 7 days of storage, respectively (Figure 3B).


Figure3. Survival of hepatitis C virus (HCV) in low void volume insulin syringes. Syringes were loaded with HCV‐spiked blood to simulate “booting.” Syringes were stored at 4°C, 22°C, and 37°C for up to 14 days before contents were flushed to infect Huh‐7.5 cells. HCV survival, a function of infectivity, was determined by the relative luciferase units (RLU) after 3 days of culture. There were 15 syringes at each time point in each experiment. A, The percentage of HCV positive syringes. B, HCV infectivity per positive low void volume syringe. Each value is the mean ± standard error of mean from at least 3 independent experiments.

The high void volume tuberculin syringes were stored for up to 63 days. In contrast to the results for the low void volume syringes, we observed a prolonged survival of HCV in the high void volume syringes at all storage temperatures; however, as with the low void volume syringes, storage at 4° was more favorable to survival of HCV than was storage at either 22° or 37°. The proportion of syringes with viable HCV declined sharply to 50% over the first 14 days of storage at 22° and 37° (Figure 4A). However, with storage at 4°, we observed a 50% decrease in viable HCV recovery only after 35 days of storage. There was a monotonic decline in the proportion of HCV‐positive syringes stored at 22° (Figure 4A). However, for syringes stored at 4° and 37°, the monotonic decline ceased after day 35. The proportion of HCV‐positive syringes recovered at the final storage duration, 63 days, was 13% at 4°, 20% at 22°, and 6% at 37°.


Figure4. Survival of hepatitis C virus (HCV) in high void volume tuberculin syringes. Syringes were loaded with HCV‐spiked blood to simulate “booting.” Syringes were stored at 4°C, 22°C, and 37°C for up to 63 days before contents were flushed to infect Huh‐7.5 cells. HCV survival, a function of infectivity, was determined by the relative luciferase units (RLU) after 3 days of culture. There were 15 syringes at each time point in each experiment. A, The percentage of HCV‐positive syringes at each time point. B, HCV infectivity per positive high void volume syringe after 1–63 days of storage. Each value is the mean ± standard error of mean from at least 3 independent experiments.

The infectivity of the HCV recovered from the high void volume syringes was determined as a function of luciferase activity. We observed at least a 90% reduction in infectivity per positive syringe after 1 day of storage at all the temperatures (Figure 4B). The infectivity of recovered virus stored at 4° tended to be higher than at other temperatures over the first 21 days of storage. At the final storage duration, 63 days, the mean infectivity per positive syringe was , , and relative luciferase units for syringes stored at 4°, 22°, and 37°, respectively. Infectivity was therefore significantly higher for syringes harboring viable virus at 4° than at 22° and 37°.

DISCUSSION

In our experimental simulation of IDU injection practices, we observed that HCV survived in HCV‐contaminated syringes for up to 63 days in high void volume syringes. Our finding supports our hypothesis that the efficient transmission of HCV among IDUs may be partly due to the ability of the virus to remain viable in contaminated syringes for prolonged periods. Moreover, we found that HCV survival was dependent on syringe type, time, and temperature. These parameters can be manipulated in the design of public health recommendations and interventions for preventing the spread of HCV among IDUs.

To our knowledge, this is the first study to establish the survival of HCV in syringes. Until recently, the absence of a sensitive tissue culture assay had made it impossible to develop suitable models to estimate HCV infectivity during the drug injection process. Lindenbach and colleagues [30, 31] recently developed a full‐length HCV genotype 2a infectious clone (HCVcc) that replicates, producing infectious virus in cell culture. To conduct the experiments, we used a genetically modified HCVcc (Jc1/GLuc2A reporter virus) virus that expresses luciferase after viral replication. After infection of Huh‐7.5 cells, the GLuc2A enzyme and infectious reporter virus are released into the culture medium. This allowed us to use the relative luciferase activity to determine HCV infectivity and HCV survival in syringes. Phan et al [29] previously demonstrated that GLuc2A expression was dependent on HCV replication, and Gluc2A expression correlated positively over time with the level of intracellular HCV RNA using quantitative RT‐PCR.

We observed that HCV survival is dependent on the type of syringe; syringes with detachable needles (high void volume) appear far more likely to transmit HCV. This observation is consistent with experimental studies in HIV [36] and epidemiologic studies in HIV and HCV, providing evidence that the probability of transmission is associated with viral burden (ie, a function of viral load and volume of inoculum) [22, 40–42]. In a recent study, Zule et al [41] found an independent association between a history of sharing high void volume syringes and the prevalence of HIV and HCV among IDUs in North Carolina. Interestingly, the investigators likened the protective role played by the use of low void volume syringes to that of male circumcision and antiretroviral therapy in reducing HIV transmission [43, 44]. The type of syringe used by IDUs depends on locality and individual preference. IDUs in the United States predominantly use fixed‐needle insulin syringes (low void syringes); pharmacies no longer sell detachable insulin syringes [40]. In areas where injection practices require volumes of water >1 mL, IDUs frequently resort to the use of syringe volumes >1 mL [41]. Interestingly, syringe exchange programs often stress the importance of providing IDUs with syringes that they prefer and meet their needs [45]. Our finding suggests the use of low void volume syringes should be stressed by syringe exchange programs to reduce HCV transmission.

The infectivity of HCV, in both low and high void volume syringes, declined sharply over the first few days. This was consistent with the observed biphasic decay rate of HCV at room temperature. Our finding of decay in infectivity of extracellular HCV is consistent with previous reports [46]. The survival of HCV in the low void volume syringes had an inverse relation to the storage temperature in many but not all conditions tested. Lower temperatures preserved the viability of HCV in the low void volume syringes to a greater extent than it did in the high void volume syringes. With the high void volume syringes, the infectivity was comparable at all temperatures after the first 14 days of storage. The time course of HCV survival in low void volume syringes is consistent with previous studies with HIV, although HCV appears to survive longer than HIV in high void volume syringes [36]. This raises the question whether the disproportionally high prevalence of HCV in comparison to HIV among IDUs could be partly explained by the differences in survival of these viruses in syringes. HCV has been shown by blood bank services to be stable for at least 7 days in plasma and serum stored at 4°C [47]. Although consistent with our finding of HCV survival in small void volume syringes stored at 4°C, these studies used PCR detection of HCV RNA, which is not a direct demonstration of viable virus. Is it possible that the duration of survival of our laboratory clone differs substantially from the duration of survival of common strains of HCV? Yes, but there is no currently available method to determine this, because there is no tissue culture system that can assess the replication or infectivity of HCV isolates from HCV‐infected individuals. Furthermore, the fact that the prevalence of HCV consistently surpasses that of HIV in IDU populations may be attributed to higher viral titer and a different range of cells susceptible to HCV infection. HIV‐1 requires entry into activated CD4+ cells for productive infection, whereas HCV needs only to enter hepatocytes, which the virus is likely to encounter because injection of HCV‐contaminated drugs brings virus to the liver on its first pass through the circulatory system. Clearly, additional research is needed to elucidate those factors that result in the higher transmissibility of HCV because virus viability alone does not seem to explain this difference.

Interestingly, harm reduction programs have effectively reduced the incidence of HIV but not HCV among IDUs [8, 24–26]. Our findings have implications in the design of public health recommendations for preventing the spread of HCV among IDUs. Successful syringe exchange programs have reduced circulation time of used syringes from 23.5 days to <3 days [26, 39]. Thus, HCV in contaminated syringes may still be viable and hence transmissible throughout the circulation time of syringes found, once syringe exchange programs are established. The types of syringes used by IDUs vary from place to place and usually depend on availability, local preferences, and cultural practices [41]. Although most IDUs in developed countries use low void volume syringes, there are still individuals who prefer high void volume syringes, and syringe exchange programs provide syringes according to an individual’s preference. In some places where homemade drugs are more common (eg, the former Soviet Union), the weaker drug solutions made in this way encourage the use of larger volume syringes that almost invariably come with detachable needles [48]. Because replacing the larger volume syringes is not practical, control of HCV transmission will require substantially expanded access to sterile syringes for IDUs in these regions.

Our study has some limitations. First, the simulation of the survival of HCV in syringes under laboratory‐controlled conditions may not accurately reflect the natural transmission dynamics among IDUs. Second, the data come from the use of a genetically modified HCV laboratory clone derived from a genotype 2a virus. Third, the spiking of HCV‐seronegative blood might not have sufficiently replicated the biological factors (eg, the presence of anti‐HCV antibodies, immune complexes, or cytokines) present in the blood of HCV‐infected individuals that could moderate HCV transmission and infectivity. However, the consistency of our results with previous epidemiologic studies that reported high HCV prevalence among IDUs supports our findings [22, 40–42].

This is the first study to our knowledge to establish the survival of HCV in contaminated syringes and the duration of potential infectiousness. The finding of prolonged duration of survival of HCV in syringes is a public health concern and adds additional evidence of the need for effective syringe exchange programs and other mechanisms to expand syringe access for IDUs.

Acknowledgments
We thank Ginger Dutschman and Amisha Patel for their technical assistance.

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Potential conflicts of interest: none reported.

Presented in part: Part of the information was presented at the 15th International Symposium on Hepatitis C Virus and Related Viruses, San Antonio, Texas, 5–9 October 2008 (abstract 379) and at the 17th Conference on Retroviruses and Opportunistic Infections, San Francisco, California, 16–19 February 2010 (abstract 168).

Financial support: This study was made possible by Clinical Translational Science Award (CTSA) (grant UL1 RR024139 to E.P.) from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and the NIH Roadmap for Medical Research (NIMH) (grant 2P30MH062294 to R.H.), funding the Yale Center for Interdisciplinary Research on AIDS (CIRA), and a diversity supplement to NIDA (grant 5U01DA017387 to E.P.). The development of the Jc1/GLuc2A system involved NIH funding (grants 1K01CA107092 and 1R01AI076259 to B.D.L.).

The content of the paper is solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH.

Source
The Journal of Infectious Diseases 2010;202:000–000
© 2010 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2010/20207-00XX$15.00
DOI: 10.1086/656213

EDITORIAL COMMENTARY

Josiah D. Rich and Lynn E. Taylor

Brown Medical School and Miriam Hospital Immunology Center, Brown University, Providence, Rhode Island

Received 1 July 2010; accepted 1 July 2010; electronically published 20 August 2010.

(See the article by Paintsil et al, on pages XXX–XXX.)

Reprints or correspondence: Dr Josiah D. Rich, The Miriam Hospital, 164 Summit Ave, Providence, RI 02906 (jrich@lifespan.org).

Hepatitis C virus (HCV) infection is a staggering problem in the United States and worldwide. In the United States, HCV is responsible for 12,000 deaths each year, is the most common bloodborne pathogen, and is a leading cause of liver transplantation. Although more than 4 million people in the Unites States and 180 million worldwide (3%) are chronically infected, most are not aware of their diagnosis. The disease burden and mortality from HCV infection are predicted to increase in the United States 2‐fold to 3‐fold over the next 10–20 years as the number of persons with long duration of infection grows. This will greatly affect individual and public health and will lead to a substantial economic burden as well. Most HCV‐related mortality is occurring in men <60 years of age (and disproportionately among non‐Hispanic black men [1]), which makes HCV a leading infectious cause of years of potential life lost. Death due to HCV infection is the most frequent cause of non–AIDS‐related death for human immunodeficiency virus (HIV)–infected persons with access to highly active antiretroviral therapy [2].

Since the discovery of a reliable test for HCV antibodies in 1990 [3], we have learned a great deal about the virus—that it leads to chronic infection in 85% of exposures, that those who are infected have an average chance of 20% of developing cirrhosis after 20 years, and that those who consume alcohol, as well as those who are coinfected with HIV, are much more likely to progress to cirrhosis and death than others. Although HCV is curable, and antiviral HCV treatment leading to viral eradication reduces liver‐related morbidity and mortality, treatment with pegylated interferon plus ribavirin is burdensome, toxic, expensive, and ineffective for half of those who attempt therapy [4–6]. Treatment initiation rates are low across varied settings [7–11]. Most patients diagnosed with chronic HCV infection have not received antiviral therapy. This is due, in part, to restrictive treatment criteria excluding patients with concomitant substance use that led to the infection in the first place [12]. The treatment landscape is on the verge of a paradigm shift with the impending launch of specifically targeted antiviral therapy for HCV (STAT‐C) to inhibit HCV‐specific enzymes. Along with higher anticipated cure rates will come higher costs of therapy, increased toxicity, thrice daily pill dosing, and the introduction of resistance. The lack of an HCV vaccine and limitations of treatment highlight the imperative of developing strategies to prevent HCV transmission.

Although perinatal and sexual transmission occur (including sexual transmission among HIV‐infected men who have sex with men [13]), the HCV epidemic is predominantly driven by the injection of illicit drugs [14]. Before 1992, when widespread screening of the blood supply began in the United States, HCV was also commonly spread through blood transfusions and organ transplants. Testing of blood donors for HCV RNA by means of nucleic acid amplification was introduced in the United States as an investigational screening test in mid‐1999 to identify donations made during the window period before seroconversion [15]. In the United States, iatrogenic transmission has been almost completely eliminated with screening of the blood supply. However, there are still incidents of transmission, such as the 2007 HCV outbreak at a freestanding private endoscopy clinic in Nevada, resulting from reuse of syringes and use of single‐use medication vials on multiple patients [16, 17].

Outside of the Unites States, one of the worst iatrogenic outbreaks of HCV infection occurred in Egypt where, from the 1960s to the 1980s, a mass campaign to eradicate schistosomiasis using repeated intravenous antischistosomal therapy inadvertently infected a generation. Decades later, the overall prevalence of HCV antibody is 15%–20% of the general population [18]. Clearly, the intravenous aspect of this campaign markedly increased the transmission rate. In the absence of mass treatment campaigns using intravenous medication, similar outbreaks are not anticipated. However, acute HCV infection is typically clinically silent, and routine screening for HCV is not recommended or done, so iatrogenic transmission of HCV may be more common than we know.

During intravenous injection by injection drug users (IDUs), blood (and any bloodborne virus such as HCV) is typically drawn up into the syringe to locate the vein, thus contaminating the inside of the syringe and creating an effective tool for transmission of whatever bloodborne pathogen is then lining the syringe. IDUs often lack knowledge about safe injection practices and the need for sterility and also often lack the necessary tools (ie, sterile syringes, diluent, mixing containers [cookers], and filters [cotton]) to prevent viral transmission.

Injection drug use remains a hidden and stigmatized behavior. Addiction is a chronic relapsing disease that is highly treatable, although most people do not get the treatment that they need. Preventive efforts should focus on IDUs, but this has not transpired on the massive scale required. IDUs have been overlooked in part because of the challenges involved in working with this population, the difficulty in finding IDUs, and the underlying stigma. In the United States, society’s major response to addiction in terms of resources has been to criminalize and further drive underground the behavior, often alienating people from treatment, which then leads to negative health and social consequences. Most IDUs are incarcerated at some point, and this has contributed to an unprecedented rate of incarceration. This “intervention” is expensive and ultimately ineffective. However, while we work to redirect policies and resources toward evidence‐based prevention, treatment, and harm reduction, mass incarceration allows us to find IDUs and provides opportunities to address prevention, diagnosis, and treatment of addiction and HCV infection in the correctional setting.

HCV is much more prevalent than HIV among IDUs, and yet the reason for this has not been fully elucidated. Are key determinants the differences in viral viability in syringes, the concentration of the virus, the volume of blood remaining in the syringes, or other factors? The increased prevalence of HCV among IDUs certainly contributes to the difference (with a higher prevalence of HCV, a given syringe is more likely to have been used by someone infected with HCV than someone with HIV) but probably does not explain it completely. Answering these questions has, until very recently, been hampered by the inability to culture HCV and the lack of a small animal model of HCV transmission.

In this issue of the Journal, Paintsil et al [19] have contributed to our understanding of the biological mechanisms of HCV transmission by developing an experimental model of injection drug use by using cultured virus from HCV‐contaminated syringes. This group is the first to our knowledge to develop a microculture assay to detect viable HCV in small volumes of blood found in syringes. This work suggests that the duration of survival of HCV in used syringes and the amount of residual blood inside the syringe are important parameters for understanding transmission. The ability to use cultured virus to explore transmission mechanisms and develop prevention interventions has the potential to revolutionize the field. There has already been an improved understanding of the role played by biocides for HCV treatment [20].

Heimer’s earlier work employed similar methods to illuminate the transmission dynamics of HIV during injection drug use [21, 22]. His group demonstrated that HIV can survive in a syringe for months. This finding, along with his team’s elegant mathematical modeling studies, proved that reducing syringe circulation time could lead to reduced HIV transmission. This provided critical early evidence in support of needle exchange programs (NEPs). NEPs have had a huge impact on the reduction of HIV transmission. However, NEPs do not seem to have had as dramatic an effect on the reduction of HCV transmission among IDUs; Heimer's HCV transmission model may facilitate understanding of why this is so. Furthermore, although noninjection drug use has been epidemiologically linked to HCV transmission (perhaps through the use of contaminated drug‐sniffing implements [23, 24]), it is possible that noninjection drug use is really just a marker for illicit, undetected injection drug use. Using simulated laboratory studies with cultured virus should contribute to understanding the mechanism(s) and relative contribution of noninjection drug use to HCV transmission.

It is estimated that an individual IDU injects 1000 times a year. HCV transmission remains unrestrained among IDUs, with incidence rates ranging from 16% to 42% per year [25]. This novel investigation provides robust evidence about the dynamics of viral transmission with syringes, using simulated injecting practices. More importantly, the ability to culture HCV heralds a new era in which the combination of basic laboratory, epidemiologic, and ethnographic research should allow a much more precise understanding of HCV transmission and pave the way for designing and targeting future public health interventions to prevent HCV infection.

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Potential conflicts of interest: L.E.T. has consulted for Vertex, is on the Speakers’ Bureau for Genentech, and has received grant support from Roche.

Financial support: Partial support for this work was provided by the National Institute on Drug Abuse (NIDA) and the National Institutes of Health (NIH) (grant K23DA020383 to L.E.T. and grants K24DA022112, P30DA013868, and T32DA13911 to J.D.R.) and the NIH Center for AIDS Research (grant P30‐AI‐42853 to J.D.R. and L.E.T.).

The content is solely the responsibility of the authors and does not necessarily represent the official views of NIDA or the NIH.

Source
Posted
August 22nd, 2010

(PhysOrg.com) — Virginia Commonwealth University researchers have identified a gene that plays a key role in regulating liver cancer progression, a discovery that could one day lead to new targeted therapeutic strategies to fight the highly aggressive disease.

Hepatocellular carcinoma, HCC, or liver cancer, is the fifth most common cancer and the third leading cause of cancer deaths in the world. Treatment options for HCC include chemotherapy, chemoembolization, ablation and proton-beam therapy. Liver transplantation offers the best chance for a cure in patients with small tumors and significant associated liver disease.

In the study, published online in the February issue of the Journal of Clinical Investigation, researchers reported that the astrocyte elevated gene-1, AEG-1, plays a key role in regulating HCC in series of cellular models. By examining human liver tumor cells of patients with HCC, the team found that the expression of AEG-1 gradually increases as the tumor becomes more and more aggressive. Using microarray technology, they analyzed cDNA from the tumor cells and determined that AEG-1 modulates expression of genes relevant to the progression of HCC, including invasion, metastasis, resistance to chemotherapy, the formation of new blood vessels, and senescence. cDNAs are complementary DNAs that are generated from mRNAs to analyze gene expression profiles.

“AEG-1 also activates multiple intracellular signaling pathways that are known to be involved in HCC progression. So, strategies to inhibit AEG-1 that could lead to the shutdown of these pathways, either by small molecules or by siRNAs, might be an important therapeutic modality for HCC patients,” said principal investigator Devanand Sarkar, Ph.D., MBBS, assistant professor in the Department of Human and Molecular Genetics in the VCU School of Medicine, and Harrison Endowed Scholar in Cancer Research at the VCU Massey Cancer Center.

siRNAs are small inhibitory RNAs that can specifically inhibit targeted mRNA and protein production. siRNAs may be used in the future for targeted inhibition of AEG-1 in patients, Sarkar said.

According to Sarkar, the team found a significantly higher expression of AEG-1 protein in more than 90 percent of tumor samples from HCC patients compared to normal human liver cells.

“The expression of AEG-1 protein gradually increases as the disease becomes more aggressive. No other genes have been shown to be upregulated in such a high percentage of HCC patients,” said Sarkar.

Further, he said that findings from a separate pool of 132 HCC patients revealed significant overexpression of AEG-1 mRNA compared to normal liver. In a subset of these patients, the team detected an increased number of copies of the AEG-1 gene.

“We observed an increase in AEG-1 DNA, mRNA and protein in HCC patients, which indicates a significant involvement of AEG-1 in HCC progression. Stable overexpression of AEG-1 converts non-tumorigenic human HCC cells into highly aggressive vascular tumors and inhibition of AEG-1 abrogates tumorigenesis by aggressive HCC cells,” he said.

Previous studies suggest that the expression of AEG-1 is very low in normal cells or tissues such as breast, prostate and brain. However, in cancers of the same organs, expression of AEG-1 is significantly increased.

The team will conduct studies to further understand the molecular mechanisms by which AEG-1 works and identify other proteins with which it interacts.

More information: A copy of the study is available for reporters at http://www.jci.org/articles/view/36460.

Provided by Virginia Commonwealth University

Source: PhysOrg – Gene Expression

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HIV-resistant cells work in mice. Can they help humans?

Paula Cannon, a biology professor at USC's Keck School of Medicine, inspects a mouse that will be infected with HIV. (Allen J. Schaben, Los Angeles Times / July 16, 2010)

By Rachel Bernstein, Los Angeles Times
August 21, 2010 6:44 p.m.

California scientists, boosted by stem cell research funding enabled by Proposition 71, are aiming for clinical trials involving gene therapy through bone marrow transplants

Clad in a yellow gown, blue foot covers, hair net, face mask and latex gloves, Paula Cannon pushed open the door to the animal room. "I hate this smell," she said, wrinkling her nose.

The stink came from scores of little white mice scurrying about in cages. Some of the cages were marked with red biohazard signs, indicating mice that had been injected with HIV.

Yet, in some of the animals — ones with a small genetic change — the virus never took hold.

Like mouse, like man? Maybe so.
 
In early 2007, a patient in Berlin needed a bone marrow transplant to treat his leukemia. He was also HIV positive, and his doctor had an idea: Why not use the marrow from one of the rare individuals who are naturally resistant to HIV and try to eradicate both diseases at once?

It worked. Sixty-one days after the patient's transplant, his virus levels were undetectable, and they've stayed that way.

Since news of the man's cure broke, HIV patients have been telephoning doctors to ask for bone marrow transplants. But it's not that simple. The treatment is too risky and impractical for widespread use.

"A bone marrow transplant — it's a horrible process you would not wish on your worst enemy unless they needed one to save their life," said Cannon, a biology professor at USC's Keck School of Medicine. There are grueling treatments to prepare a patient for transplant; the danger of rejecting the marrow; and the risk of graft-versus-host disease, wherein the marrow attacks the patient.

And that's assuming the patient can find a matching donor — a difficult proposition in itself — with the right HIV-resistant genetic constitution, which is present in only about 1% of the Caucasian population.

But there could be another way.

Instead of sifting through the sands for a rare donor and then subjecting a patient to the dangers of a bone marrow transplant, Cannon and her colleague Philip Gregory, chief scientific officer at the Richmond, Calif.-based biotech company Sangamo BioSciences, began to think: They could use gene therapy instead, to tweak a patient's own cells to resistance — and recovery.

The mouse "cure," they say, suggests they're on the right track.

Now, with $14.5 million from the California Institute for Regenerative Medicine, the San Francisco-based stem cell research-funding center created by 2004's Proposition 71, Cannon, Gregory and researchers at the City of Hope cancer center in Duarte are working toward bringing the technique to clinical trials within four years.

Cannon and other HIV researchers insist that, despite cancers and deaths associated with past gene therapy trials, it's the right way to target the disease. They cite recent successes, including treatments that cured children with the "bubble boy" syndrome and helped blind children regain their vision.

"I don't think anyone would want to do gene therapy if there were an alternative," said Caltech biologist David Baltimore, one of the many L.A.-based researchers pursuing gene therapy strategies to prevent or cure HIV. "I think it's absolutely necessary. Nothing else will work."

Since AIDS emerged in the early 1980s, development of anti-HIV medications has turned the disease from a virtual death sentence into a chronic, manageable condition.

But the clamor for a cure hasn't quieted.

Vaccine trials have failed; drug-resistant strains are on the rise; and the meds, which can have uncomfortable side effects such as fatigue, nausea and redistribution of body fat that creates a so-called buffalo hump, cost about $20,000 a year.

A bone marrow transplant is about five times as expensive, but it would have to be done only once.

The question was, could researchers create bone marrow stem cells that — just like the marrow the Berlin patient received — lack the crucial gene, CCR5, that normally lets HIV into the key immune cells it destroys?

In 2006, Gregory asked Cannon if she was interested in testing whether a tool his company developed, called a zinc finger nuclease, could do the trick.

Zinc finger nucleases are genetic scissors, cutting DNA at a specific site — say, in the middle of the CCR5 gene. When the cell glues the gene back together, it usually makes a mistake, resulting in a gene that no longer works.

"It just jumped out at me as, 'Oh my gosh, that's actually something that could work,' " Cannon said.

The team spent about a year optimizing the procedure for treating delicate stem cells with the CCR5 snippers.

They tested the method using so-called humanized mice — ones engineered to have a human immune system — because HIV doesn't infect normal mice. When stem cells were treated with the molecular scissors before being injected into mice, the resulting immune system lacked CCR5, exactly as the scientists had hoped.

These mice acted just like the Berlin patient — they fought off the virus.

Ready to make the leap from mouse to man, Gregory found a third leg for the team: researchers at City of Hope, who had extensive bone marrow transplant expertise.

"They brought Paula's data to us and we said, 'Wow, this looks fantastic,'" said Dr. John Zaia, City of Hope's deputy director for clinical research.

Researchers there are now working toward clinical trials, optimizing every element of the treatment for safety, effectiveness and reproducibility.

On a wiltingly hot afternoon in July, lab manager Lucy Brown maneuvered a computer mouse across three screens speckled with red, yellow and green dots.

The computer was hooked to a flow cytometer — a collection of black boxes, green wires and silver knobs that can detect subtle differences between cells and separate them at a rate of 50,000 per second. This is how the scientists will separate stem cells from patients' blood once trials are underway, to be sure that the genetic fix in the CCR5 gene was made, and kept.

Upstairs, machines with mazes of sterile tubes and pumps stood ready to prepare cells for CCR5-snipping. Here, the scientists will purify the bone marrow stem cells, increasing their numbers first to 5% of total cells, up from a measly 0.1% in the starting mixture, and then to 99%. At this point they can begin testing methods to clip the cells' DNA.

When all is perfected, the scientists will have a precise recipe for producing batches of engineered stem cells, including exactly how long the cells should be treated, how much of each chemical needs to be added, how pure the cells need to be, and thousands of other details.

"We are literally writing the book on how you do this," said David DiGiusto, director of City of Hope's bone marrow stem cell therapy research.

To receive FDA approval for clinical trials — a goal they hope to achieve in three to four years — the researchers must prove that they can safely and reliably prepare the cells. Once they get the green light, the first cases will probably be people like the Berlin patient who need bone marrow transplants to treat AIDS-related lymphoma.

They'll modify the patients' cells in the stringently sterile manufacturing lab that DiGiusto designed with details such as cove molding and seamless floors so there are no corners or cracks to collect dust. Anyone who enters must wear a full bunny suit, much like the one Cannon wears in her mouse room, to keep from contaminating the delicate cells.

Some have advertised the effort as a quest for the elusive "C" word, but Cannon doesn't quite see it that way.

"People say we're trying to cure HIV," she said. "I think of it more as, we're just trying to make the body live quite happily and healthily with a small amount of virus."

rachel.bernstein@latimes.com
 
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