Asthma is a disease that affects all ages, races and ethnic groups. Its incidence is increasing both in Westernized countries and underdeveloped countries. It involves inflammation, genetics and environment and therefore, proteins that exacerbate the asthmatic, allergic phenotype are important. Our laboratory purified and cloned a histamine releasing factor (HRF) that was a complete stimulus for histamine and IL-4 secretion from a subpopulation of allergic donors' basophils. Throughout the course of studying HRF, it was uncovered that HRF enhances or primes histamine release and IL-13 production from all anti-IgE antibody stimulated basophils. In order to further delineate the biology of HRF, we generated a mouse model.We constructed an inducible transgenic mouse model with HRF targeted to lung epithelial cells, via the Clara cells. In antigen naïve mice, overproduction of HRF yielded increases in BAL macrophages and statistical increases in mRNA levels for MCP-1 in the HRF transgenic mice compared to littermate controls. In addition to demonstrating intracellular HRF in the lung epithelial cells, we have also been able to document HRF's presence extracellularly in the BAL fluid of these transgenic mice. Furthermore, in the OVA challenged model, we show that HRF exacerbates the allergic, asthmatic responses. We found statistically significant increases in serum and BAL IgE, IL-4 protein and eosinophils in transgenic mice compared to controls.This mouse model demonstrates that HRF expression enhances allergic, asthmatic inflammation and can now be used as a tool to further dissect the biology of HRF. We identified a histamine releasing activity that was found in late phase fluids from nasal lavages, bronchoalveolar lavage (BAL) fluids and skin blister fluids that directly induced histamine release from basophils isolated from a subpopulation of allergic donors Plasmodium falciparum, Wucheria bancrofti, Brugiia malea and Schistosoma Mansinai, all of which possess mast cell/basophil histamine releasing activity Homologs of HRF have been described in parasites including in vitro, methylcholine sensitivity and symptoms of asthma were highly correlated in vivo, it may be contributing to mediator release that is found in the late response. Further understanding of the biology of HRF may help explain the varying severities of allergic disease.The importance of the association of HRF with human allergic disease has been previously documented in numerous publications. It should be noted that while these observations were made with crude HRF, the same exists with recombinant material. For instance, HRF has been found in human respiratory secretions (BAL) and skin blister fluids Although HRF has been extensively investigated for many years, most studies have been carried out in primary human or cultured cells. Currently, there is no established animal model available to further explore the function of HRF. One group from Taiwan generated HRF knockout mice by targeted gene disruption All procedures performed on mice were in accordance with the National Institutes of Health guidelines for humane treatment of animals and were approved by the Johns Hopkins University Institutional Animal Care and Use Committee (ACUC). The ACUC protocol number is MO07M197.The HRF transgenic plasmid was generated by the combination of three main components. The first component is the pTRE-tight vector, which contains a modified TRE (tet response element) controlling the inducible expression of the gene of interest. The second component is human HRF cDNA, which was cloned from U937 cells by RT-PCR and confirmed by sequencing. The third component is the pIRES2-EGFP vector . The IRES2  allows the enhanced green fluorescent protein (EGFP) gene to be expressed individually as a reporter protein along with HRF in order to facilitate the recognition of expression of transgenic human HRF.Transgenic HRF mice were generated by pronuclear injection of the transgenic plasmid described above by the John Hopkins Transgenic Core. HRF transgenic mice were crossbred with CC10-rtTA mice on a C57BL/6 background to obtain double transgenic CC10-rtTA/HRF mice (transgene++) for the functional experiments. All experiments shown in these studies were completed during crossbreeding to reach pure background at generation 10. Therefore each protocol was completed with littermate control mice to account for generational variety. Mice were used at 6–11 weeks of age. All mice were housed in cages with microfilters in a specific pathogen-free environment.5′-GAC GTA AAC GGC CAC AAG TT-3′; EGFP-R, 5′-GAA CTC CAG CAG GAC CAT GT-3′; TRE-HRF-F, 5′-GTG TAC GGT GGG AGG CCT AT-3′; TRE-HRF-R, 5′-GTT TCC TGC AGG TGA TGG TT-3′; and CC10-F, 5′-ACT GCC CAT TGC CCA AAC AC-3′; CC10-R, 5′-AAA ATC TTG CCA GCT TTC CCC-3′. The following PCR protocol was used: 95°C for 5 min; 30 cycles of 95°C for 45 sec, 62°C (EGFP and HRF) or 60°C (CC10) for 45 sec, and 72°C for 45 sec; and a final extension at 72°C for 5 min.The presence or absence of the transgene in the resulting animals and their progeny was determined using tail DNA and PCR analysis. The primer sets used for PCR were EGFP-F, Induction of transgene overexpression-Protocol IAll TRE-HRF-EGFP transgenic mice and CC10 control mice were maintained on normal water until they were 6 weeks old. Then doxycycline (Dox) water at 1 mg/ml in 4% sucrose kept in dark bottles to prevent light-induced degradation was administrated for 3–4 weeks. Regular drinking water was given to littermate controls for comparison for the duration of the experiment (n = 4−11 per group).Ovalbumin (OVA) or PBS sensitization followed by OVA challenge-Protocol IIThe sensitization and challenge protocol was completed as previously described OVA sensitization followed by OVA (or PBS) challenge-Protocol IIITo further explore the role of HRF in the allergic diathesis we design additional experiments that compared effects of HRF on mice that are sensitized with OVA and then challenged with OVA (as previously described in 2) or with sham challenge of PBS (n = 6−8 per group). The protocol was otherwise the same as previously described.Airway hyperresponsiveness (AHR) was assessed by methylcholine-induced airflow obstruction from conscious unrestrained mice placed in a whole body plethysmograph  Lung tissues and BAL samples were obtained as previously described 3VO4, and proteinase inhibitor cocktail, BD BioSciences, San Jose, CA) and then centrifugated at 10,000×g at 4°C for 30 min to obtain the cellular proteins in the supernatant. The protein concentrations were determined by BCA Protein Assay , and 20 µg of total protein from each sample were boiled for 5 min and electrophoresed on 4–20% Tris-Glycine gels , transferred to nitrocellulose membranes, and blocked in blocking buffer  for 1 hr at room temperature. The membranes were blotted with anti-EGFP  or anti-HRF  and embedded in paraffin. The lung tissue sections (5 µm in thickness) were deparaffinized with xyline, rehydrated gradually with graded alcohol solution , and then washed with deionized water and immersed in 3% BSA for 1 h to block nonspecific binding. These slides were then incubated with primary mouse anti-HRF antibody at dilutions of 1∶200 for 18 h at 4°C, washed twice in PBS/Tween-20 solution, incubated with a Texas Red-conjugated secondary antibody for 1 h at room temperature, and photographed with a fluorescent microscope. We chose anti-HRF antibody to directly measure HRF. Therefore, an anti-GFP antibody was unnecessary.Lung tissues were fixed in Protocol Safefix II (Fisher Scientific Co.), and processed by AML Laboratories . Briefly, samples were embedded with paraffin, sectioned at 5 µm, deparaffinized, dehydrated, and stained with hematoxylin and eosin (H&E). In collaboration with Dr. Allen Myers, Head of the Johns Hopkins Histologic Core, these specimens were examined under bright-field optical microscopy using a light microscope, and corresponding digital images were captured for subsequent analysis by a Spot CCD Camera driven by Advanced Spot RT Software version 3.3 (Diagnostic Instruments Inc.).5′-GAC GTA AAC GGC CAC AAG TT-3′; EGFP-R, 5′-GAA CTC CAG CAG GAC CAT GT-3′; actin-F, TCC TGT GGC ATC CAG GAA ACT; actin-R, GGA GGA ATG ATC CTG ATC TTC; IL-4-F, 5′-TCA TCG GCA TTT TGA ACG AG-3′; IL-4-R, 5′-GAA TCC AGG CAT CGA AAA GC-3′; IL-13-F, 5′-TCA GCC ATG AAA TAA CTT ATT GTT TTG T-3′; IL-13-R, 5′-CCT TGA GTG TAA CAG GCC CAT TCT-3′; MCP-1-F, 5′-ACC AGC CAA CTC TCA CTG AAG C-3′; MCP-1-R, 5′-CAG AAT TGC TTG AGG TGG TTG TG-3′; MDC-F, 5′-CCT GGT GGC TCT CGT CCT TC-3′; MDC-R, 5′-CAG GGG ATG GAG GTG AGT AA-3′) under formulated conditions. Amplified PCR products were analyzed by means of electrophoresis, and the intensity of the bands and the ratio of specific mRNA to β-actin were analyzed with the Bio-Rad Gel Doc system and the Quantity 4.4.1 software (Bio-Rad Laboratories).Lungs were rapidly dissected and frozen on dry ice. For extraction of total RNA, tissues were homogenized in 1 ml of ice-cold TRIzol reagent (Invitrogen) and the total RNA was reverse-transcribed into cDNA using a random hexamer and a GeneAmp RNA PCR Kit . PCR analysis was performed by AccuPower PCR PreMix kit  with each amplified primer set  according to the manufacturer's instructions . The levels of OVA-specific IgE in serum were determined using a commercial ELISA per the manufacturer's instructions . IL-4 and eotaxin levels of BAL fluids were quantitated using commercial ELISA kits  according to the manufacturer's instructions.t test. A p value of less than 0.05 was considered statistically significant.All experiments were repeated with multiple mice and matched littermate controls as indicated by the n values in each experiment. The quantitative data of continuous variables were expressed as mean ± S.E.M of each group. The statistical difference between experimental groups was determined by Student's The schematic of the physical map of the transgene is shown in After crossbreeding, both transgene++ and CC10 control mice were treated with Dox at 1 mg/ml for 3–4 weeks. Regular drinking water was also given to littermate controls for comparison. Transgene expression was verified by both Western blot and mRNA analysis of lung tissue. First, lung tissue lysates were analyzed by Western blot for both EGFP and HRF expression, as shown in et al found that in vivo administration of HRF increased total and Ag-specific Ig synthesis, but these studies were done in the more conventionally allergic BALB/c mice BAL cells were examined and there was a significant increase in total cell counts in transgene++ mice compared to CC10 mice following dox induction and the change was primarily due to an increase in macrophages  and PBS (p = 0.05) sensitized groups , Panel BAsthma is a chronic inflammatory disorder of the lung and TH2 inflammation of the airway is a major component of asthma. It has been demonstrated in animal models of asthma that allergens can elicit TH2 inflammation and that IL-4 is essential in this response. For example, in the presence of IL-4, naïve CD4+ T cells differentiate into TH2 cells Airway physiology abnormalities are a hallmark of asthma in human and animal models of asthma. Methylcholine challenge was used to measure airway reactivity. We saw an increase in airway reactivity following sensitization and challenge with OVA, however, there were no significant differences between littermate controls, except at the top dose (50 mg/ml) between CC10/OVA vs. CC10/PBS sensitized (data not shown). The over expression of HRF either by Dox induced over expression, or by natural allergen induced up-regulation did not appear to significantly effect the airway reactivity to methylcholine compared to that seen with control CC10 mice. This result is not unexpected since it is known that AHR is IL-13 dependent To further explore the role of HRF in the allergic diathesis we designed additional experiments that compare effects of HRF on mice that are sensitized with OVA and then challenged with OVA (as previously done in Protocol II) or with sham challenge of PBS (n = 6−8 per group). The absence of OVA challenge would further dissect the role of HRF in the sensitization and response phases. The protocol and phenotypic analysis were otherwise the same as previously described.Transgene expression was confirmed in each mouse as previously described and HRF protein was up regulated following Dox administration on transgene++ mice compared to CC10 littermate controls (p = 0.03). Of note again, is that HRF protein levels are increased following OVA sensitization and challenge compared to OVA sensitized PBS challenged litter mate controls in both transgene++ (p = 0.03) and CC10 mice . This indicates that OVA challenge increases naturally occurring HRF, not unlike the results in Protocol II , Panel AOverall, results seen in the Protocol III were similar to the data shown in the previous Protocol II between transgenic++ and CC10 control mice OVA sensitized and challenged. Namely, an increase in airway reactivity to methylcholine challenge following sensitization and challenge with OVA was seen, however, there were no significant difference between littermate controls between transgene++ OVA versus PBS challenge. Also, IL-4 levels and eosinophil numbers were elevated (data not shown). However, total cells and macropahges were not significantly increased as seen in both Protocol I, which are antigen naïve mice, and Protocol II which were not sensitized with antigen. These data indicate that HRF does not affect macrophage recruitment during OVA sensitization, but HRF may initiate inflammation via macrophage recruitment in the absence of sensitization.As expected, serum IgE levels significantly increased following OVA sensitization and challenge in both the CC10 controls and the transgene++ group when compared to littermate controls challenged with PBS , Panel AIn our transgenic HRF mouse model, we observed an induction of HRF in the lung and corresponding increased numbers of macrophages as well as levels of MCP-1 levels in the lungs. These data were shown in Protocol I  and 3. MWe found that HRF levels in the lungs were increased by both OVA sensitization and challenge. This increase was observed in both HRF transgenic mice and CC10 control mice following either OVA sensitization or OVA challenge see . The immin vitro, HRF has also been shown to cause chemotaxis of eosinophils and secretion of IL-8 from allergic donors We also showed after OVA sensitization and challenge that HRF transgenic mice had increased levels of the TH2 cytokine IL4, but not eotaxin, IL-5 or IL-13. IL-4 is necessary for the induction of IgE production and the increases may be responsible for the observed increase in total IgE. In addition, IL-4 has been shown to play an important role in the migration of eosinophils from the lung into the airway HRF has been shown to be present in the BAL of asthmatic patients HRF has been previously shown to activate epithelial cells HRF transgenic mice have increased total serum IgE levels following either OVA sensitization or challenge  and 11, in vitro in human studies over the past several years and is now seen in the mouse model of allergic inflammation. Future studies with this transgenic mouse model will elucidate the specific mechanisms of HRF's actions on the inflammatory process with particular emphasis on the mouse basophil.In summary, endogenous HRF is up-regulated after either allergen sensitization or challenge. Following OVA sensitization and OVA challenge, we see the same trends, namely, increases in IL-4 and eosinophils and no additional increases in AHR. However, there are no differences in total cells and increases in macropages following OVA sensitization and challenge with PBS. This points to the fact that HRF does not exert its effect in the sensitization phase alone. OVA challenge is necessary for HRF to exert a local effect in the response phase as measured by increases in BAL IgE levels. In contrast, either OVA sensitization or OVA challenge will suffice for HRF to have an effect on serum IgE levels. In general, HRF augments the inflammatory response to allergens. This has been seen


To assess the impact of vocational rehabilitation on psychopathology, social functioning and cognitive functioning in schizophrenia34 patients with DSM IV diagnosis of chronic schizophrenia were compared 40 patients with same diagnosis but not attending vocational rehabilitation using PANSS, SCARF social functioning Index and MMSE.Basic psycho-socio-demographic data were comparable in both groups except more hospitalization in the no rehabilitation group. Comparison of social functioning, cognitive functioning and psychopathology showed significant improvement in rehabilitated patients. Cognitive functioning had positive correlation with occupational role in the rehabilitated group and negative correlation in the rehabilitated group. Social functioning had negative correlation with positive and negative symptoms, general psychopathology and total PANSS score and cognitive symptoms in patients without rehabilitation.The present concludes that there is a definite limitation in the domains of social functioning, cognitive functioning and psychopathology in chronic schizophrenia patients who had no rehabilitation. However vocational rehabilitation significantly improves these limitations, which in turn help these patients to integrate into the society so as to function efficiently in their roles as parents, home makers and social beings. Schizophrenia is one of the severe forms of mental illnesses, which demands enormous personal and economic costs. Globally it is estimated that 25 million have schizophrenia. The majoRehabilitation is essential to help such patients to return to the previous level of functioning. It helps them to cope with the illness, strive for greater reliance and enhanced functioning and to improve the quality of life. Vocational rehabilitation programme utilizes the work for the improvement of symptoms, interpersonal relationships, and cognitive functioning. It brings forth significant changes in their over all-functioning level i.e., living, learning, and work related conditions. Vocational rehabilitation has been shown to improve employment rates for individuals with schizophrenia.–4 Thus, Even though rehabilitation programmes are in progress for many years in western setting it is still under developed in our country and only limited studies have been conducted in the Indian context to evaluate their effectiveness. All these observations together with the interest in the rehabilitation of patients with chronic schizophrenia prompted the investigator to study how far vocational rehabilitation reduces the symptoms and improve social and cognitive functioning of these patients.To identify and compare the level of social functioning, cognitive functioning, and psychopathology in patients with chronic schizophrenia undergoing rehabilitation with those patients not undergoing rehabilitation.To assess the correlation between cognitive functioning, social functioning, psychopathology, and social functioning in the above two groups.This study was conducted at Government Mental Health Center, Kozhikode, one of the largest state government owned mental health centers with a bed strength of 474. About 150 patients attend the outpatient department and around 10-15 patients get admitted in the family therapy ward on every day basis. This hospital has got extensive rehabilitative services. The male rehabilitation side is a well-equipped unit having facilities for school and college notebooks manufacturing, bookbinding, offset printing, carton making, medicine cover making horticulture. The female unit offers facilities for bookbinding, spinning, and medicine cover making. Both male and female rehabilitation centers work from 10.00 am to 4.00 pm with one-hour break for lunch from 1.00 pm to 2.00 pm on all government working days. Patients will be allocated appropriate job based on their aptitude and current level of functioning. These patients are paid incentives appropriate to the quality and quantity of work. On an average most of these patients are getting 75 to 100 Rupees per day.Non-probability purposive sampling technique was used for the study. The sample was selected from the out patient population as per the criteria laid out below.Clinically diagnosed chronic schizophrenia as per DSM IV Criteria with at Patients attending vocational rehabilitation for at least 6 months - Group IPatients who had no rehabilitation during life time - Group IIPatients who are able to communicate in MalayalamAssociated medical illnesses severe enough for not to participate in the studySeizure disorder and mental retardationAcute exacerbationSpecially designed proforma for documenting socio-demographic and illness data such as age, sex, marital status, education, religion, type of family, family income, total duration of illness, duration of treatment, duration of rehabilitation and the number of hospitalization.SCARF Social Functioning Index was usedMini Mental State examination to assesPositive and Negative Syndrome Scale to assesAfter getting approval from the institutional ethics committee data were collected during the period from 08-02-2006 to 21-03-06. A written informed consent was obtained from all the participants. Patients were comfortably seated, explanation about the objectives of the study was given and confidentiality of the data was ensured. First, the investigator interviewed the client for collecting socio-demographic data and social functioning. Following this MMSE was given and then PANSS was administered. Time taken for one client was 45 minutes.t-test. Correlation between cognitive functioning, social functioning, and psychopathology was performed by Pearson's Coefficient of Correlation.Socio-demographic data were analyzed using frequencies, percentage, and chi-square. Comparison of social and cognitive functioning and psychopathology was done by Tables The present study proves that vocational rehabilitation for six months improves social functioning, reduces the severity of symptoms, reduces re-hospitalizations and enhances cognitive functioning in patients with chronic schizophrenia. This finding is in concordance with the findings of many western studies.9–13et al.[Vocational rehabilitation showed significant improvement in positive symptoms, negative symptoms, thought disturbance and paranoid ideation. Mueser et al. in theiret al. have fouet al. from Banet al.[In schizophrenia, neurocognitive factors play an important role in respect of their social competence. Present study revealed a positive correlation between social functioning especially occupational role and cognitive functioning in patients who had undergone vocational rehabilitation. Same time patients without vocational rehabilitation had negative correlation of social functioning with cognitive functioning. These findings are in consistent with the findings of Bryson and Bell, Liddle, and Pennet al. found thPresent study showed significant negative correlation of social functioning with positive symptoms, negative symptoms, and general psychopathology in patients with vocational rehabilitation. This is also in accordance with the findings of Mc Gurk and Mueser and HoffBefore concluding, some of the methodological limitations of this study have to be considered. Being a smaller sample and cross sectional study with only one point assessment without baseline assessment prior to rehabilitation generalization of our findings is limited. Investigator was not blind in assessing patients with and without rehabilitation. Moreover, the investigator could not control extraneous factors like physical and psychological factors and medications, which may influence the studied variables.The present study draws the conclusion that there is a definite limitation in the domains of social functioning, cognitive functioning and psychopathology in chronic schizophrenia patients who had no rehabilitation in their life time. However, vocational rehabilitation significantly improves these limitations, which in turn help these patients to integrate into the society so as to function efficiently in their roles as parents, home makers, and social beings. Being the only source of income in many patients, vocational rehabilitation has significantly helped to earn livelihood for buying medicines as well as to look after the family. Vocational rehabilitation has also reduced the relapse rate and subsequent hospitalization which indirectly reduces the treatment cost and burden for the caregivers. An attractive feature of this type of rehabilitation is that all patients irrespective of their functional status can be accommodated to different types of rehabilitative work available in the local community. Considering the cost effectiveness and lower cost in implementation, this type of rehabilitation may be an ideal model for a developing country like India. In this context, there is a need for future studies with larger sample size and with more longitudinal and periodical assessments to assess the impact of vocational rehabilitation using locally available means.


Penile rehabilitation therapy following radical prostatectomy is a much debated topic. Erectile dysfunction is still a significant contributor to postoperative morbidity following radical prostatectomy, despite meticulous nerve-sparing technique. Secondary smooth muscle changes in the penis have been identified as the underlying causes of penile atrophy, veno-occlusive dysfunction, and fibrosis. Initial observations that intracavernous injection therapies used on a regular basis postoperatively resulted in improvements in the return of spontaneous erectile function led to the development of penile rehabilitation protocols. Chronic dosing of PDE-V inhibitors is now commonly used by urologists after radical prostatectomy. Despite the current enthusiasm of penile rehabilitation therapy, current scientific evidence with clinical trials is still limited. Erectiledysfunction (ED) continues to be a significant complaint among men undergoingradical prostatectomy (RP), despite meticulous nerve-sparing technique. Thediscrepancy between preservation of the nerves and recovery of erectile functionleads us to believe that the etiology is likely multifactorial. Smooth musclealterations and fibrotic changes in the penis following surgery were identifiedas underlying causes of penile atrophy, veno-occlusive dysfunction, andPeyronie's-like changes. Other mechanisms, such as penile hypoxia, are likelyto contribute to post-RP erectile dysfunction as well.The topicof penile rehabilitation therapy (PRT) has become an area of intense interestover the last decade. Initial observations that intracavernous injectiontherapies used on a regular basis postoperatively resulted in improvements inthe return of spontaneous erectile function led to the development of penilerehabilitation protocols . A centrThe basicscience behind the mechanism of erection has been discussed previously and isbeyond the scope of this review . With thWhile there have been many studies in the rat modelshowing the benefits of local vasoactive therapies, the crossover to clinicalsignificance in the human has been more difficult to prove see . MontorThe evidence for the use of oral agents for penilerehabilitation is even weaker. In 2005, Mulhall et al. reported in a nonrandomizedstudy that men who underwent pharmacologic penile rehabilitation following RPresulted in higher rates of spontaneous functional erections . Men whoThe benefit induced by a PDE-V inhibitorrehabilitation approach compared to an on demand PDE-V administration has notbeen confirmed. In a follow-up to their first study , MontorsP < .001),with potency rates of 86% versus 66%. The authors concluded that in cases ofearly penile erection, daily low-dose sildenafil leads to a significantimprovement in the recovery of erectile function. However, the limitations tothis study are that it was performed at a single center and 95% of patients hadnocturnal erections following catheter removal, which is not typical.Bannowsky et al. from Germany reported in 2008 theirfindings of low-dose sildenafil for rehabilitating erectile function afternerve-sparing RP . Twenty-Padma-Nathan et al. performed the first randomized,placebo-controlled trial of chronic PDE-V inhibitor therapy for the purposes ofpenile rehabilitation and presented their findings at the 2003 AmericanUrological Association annual meeting . They coA subset of these men hadnocturnal penile tumescence and rigidity (NPTR) testing performedpreoperatively and at various time points postoperatively . The autThe limitation of this study is that while being suggestive, nocturnal tumescence data interpretation does notnecessarily correlate with return of clinically usable erections.In contrast, in the largest randomized, double-blind, double-dummy, multicenter, parallel study done to date, Montorsi et al. recently reported on a vardenafil trial after bilateral nerve-sparing RP . A totalPoint-counterpoint debates have become commonregarding the efficacy of penile rehabilitation following RP. In one such example, Abraham Morgentalerfrom Harvard University listed several intriguing arguments . First, Similarly, in a more recent point-counterpointdebate at the 2008 AUA Annual Meeting in Orlando, Fla, Craig Donatuccifrom Duke University was charged with providing the contrary argument as whether penilerehabilitation was effective after RP. He presented many of the same points listed above, describing thepitfalls of the clinical trials that have been presented thus far. He stated: “There is not yet enough evidenceto declare penile rehabilitation effective; but I am not sufficiently convincedof the ineffectiveness of penile rehabilitation to recommend against it. Myopinion is that the benefits of penile rehabilitation are “not proven”; yet itis currently the standard of care” .The study of penile rehabilitation is in activeevolution. There are intriguing data supporting the use of penilerehabilitation in the animal model, but these studies may not translate intohuman effect. While many published reports of clinical trials point towardimproved outcomes with active treatment, these studies have significantlimitations. Most recently, the onlylarge, randomized, placebo-controlled trial to date showed no clear benefit ofrehabilitation with vardenafil versus on demand dosing. In fact, quite theopposite was shown. Clearly, more randomized, placebo-controlled trials areneeded. Unfortunately, these presentmany challenges. First, these trials prove costly with poor long-termcompliance and significant early dropout rates. Second, it cannot be overlookedthat differences in nerve-sparing technique and outcomes between surgeons canconfound interpretation of results. Eventhough the benefits of penile rehabilitation are still questionable, thepractice of frequent and chronic administration of PDE-V inhibitors has becomestandard of care for many urologists. We suggest proceeding with caution whenencouraging our patients to adhere to a rehabilitation regimen, until moredefinitive results are available.


As quinacrine is known to be a substrate for P-glycoprotein multi-drug resistance (MDR) transporters, we circumvented its poor BBB permeability by utilizing MDR0/0 mice that are deficient in mdr1a and mdr1b genes. Mice treated with 40 mg/kg/day of quinacrine accumulated up to 100 µM of quinacrine in their brains without acute toxicity. PrPSc levels in the brains of prion-inoculated MDR0/0 mice diminished upon the initiation of quinacrine treatment. However, this reduction was transient and PrPSc levels recovered despite the continuous administration of quinacrine. Treatment with quinacrine did not prolong the survival times of prion-inoculated, wild-type or MDR0/0 mice compared to untreated mice. A similar phenomenon was observed in cultured differentiated prion-infected neuroblastoma cells: PrPSc levels initially decreased after quinacrine treatment then rapidly recovered after 3 d of continuous treatment. Biochemical characterization of PrPSc that persisted in the brains of quinacrine-treated mice had a lower conformational stability and different immunoaffinities compared to that found in the brains of untreated controls. These physical properties were not maintained upon passage in MDR0/0 mice. From these data, we propose that quinacrine eliminates a specific subset of PrPSc conformers, resulting in the survival of drug-resistant prion conformations. Transient accumulation of this drug-resistant prion population provides a possible explanation for the lack of in vivo efficacy of quinacrine and other antiprion drugs.Quinacrine is a potent antiprion compound in cell culture models of prion disease but has failed to show efficacy in animal bioassays and human clinical trials. Previous studies demonstrated that quinacrine inefficiently penetrates the blood-brain barrier (BBB), which could contribute to its lack of efficacy  Prion diseases belong to the class of neurodegenerative disorders that include Alzheimer, Parkinson and Huntington diseases. In each of these disorders, a specific protein in the brain changes shape and accumulates, leading to neuronal loss and damage. These diseases are uniformly fatal after a period of neurodegeneration, dementia and motor dysfunction. Quinacrine, an antimalarial drug, is able to eliminate prions from dividing cells in culture, yet is ineffective in diseased mice and human patients. Here, we provide an explanation for this failure. Our data indicate that the administration of quinacrine results in the proliferation of drug-resistant prions. This insight will enable us to develop more effective antiprion therapeutics in the future. C) into an aberrantly folded conformation (PrPSc) Sc accumulation has been demonstrated to be the primary pathogenic event in prion disease, the exact molecular and cellular mechanisms of its formation and the ensuing neurodegeneration remain largely unknown.Prion diseases are a class of rare, neurodegenerative disorders that include Creutzfeldt-Jakob disease (CJD) and kuru in humans, BSE in cattle, and scrapie in sheep To date, no therapeutic agent against prion diseases has been identified. Numerous compounds have been found to have antiprion activity in cell culture models of prion disease, including pentosan polysulfate, dextran sulfate, HPA-23, Congo red, suramin, dendritic polyamines and quinacrine, among others; for a comprehensive review, see Scin vivo has not appeared to be promising. Collins and colleagues reported that the incubation time of mice intracerebrally inoculated with prions and subsequently treated with quinacrine via gavage feeding did not differ from that of untreated mice Among the antiprion compounds, quinacrine seemed to be the most promising for immediate application in the treatment of prion disease because it has been used for decades as an antimalarial drug Sc (EC50) in cell culture were reported to be ∼1 µM and ∼7 µM for extracellular and intracellular accumulation, respectively in vivo seemed likely to result from its insufficient accumulation in the brain.In wild-type mice, quinacrine inefficiently penetrates the blood-brain barrier (BBB). After an oral dose of 40 mg/kg/day, quinacrine accumulates in the brain at concentrations of less than 1 µM (MDR1[ABCB1] and MDR2[ABCB4]) and three genes in rodents  MDR1 in humans, and mdr1a and mdr1b in mice, confers the vast majority of observed multidrug resistance. Hereafter in this paper, “Pgp” will refer to the proteins encoded by this class of MDR genes (there is no current evidence that mdr2 is involved in quinacrine efflux). Given that quinacrine is a substrate for Pgp, we tested whether deletion of the mdr genes , denoted as MDR0/0) would improve the pharmacological profile of quinacrine and enhance its in vivo efficacy.The finding that the P-glycoprotein transporter is involved in the efflux of quinacrine across the BBB 0/0 mice at concentrations exceeding 100 µM. Despite attaining these high concentrations in the brain, quinacrine failed to extend the survival times of prion-inoculated MDR0/0 mice. The results suggest that the failure of quinacrine in vivo cannot be attributed solely to its pharmacokinetic properties. Quinacrine treatment transiently reduced PrPSc levels in the brains of mice, but PrPSc levels recovered during the course of treatment. Parallel results in cell culture suggest that continuous quinacrine treatment leads to the selective survival of drug-resistant prion conformations. However, these resistant conformations do not stably propagate in the absence of quinacrine.Here, we report that quinacrine can accumulate in the brains of orally treated MDR0/0. Mice were inoculated intracerebrally with 30 µL of 1% brain homogenate containing the Rocky Mountain Laboratory (RML), rodent-passaged, scrapie prion strain. An oral regimen of 40 mg/kg/day of quinacrine was initiated at selected time-points  and continued for different time intervals . The mice were monitored for neurological dysfunction and sacrificed upon the onset of prion disease. At least 9 mice were allowed to reach the endpoint of disease for each arm of the experiment and used to determine the mean incubation period . For some arms, several mice were sacrificed during the course of the experiment in order to measure quinacrine uptake, PrPSc levels, and neuropathologic changes (see below). Treated mice will be referred to as S[a-b] or S[a>], for which “S” is the mouse strain, “a” indicates the initiation of quinacrine in dpi, “b” indicates the cessation of treatment in dpi, and “>” indicates lifelong treatment.We evaluated whether quinacrine treatment would extend the survival time of prion-infected mice of three genetic backgrounds: CD1 (wild-type outbred), FVB (wild-type inbred) and MDR. Short-term quinacrine treatment (≤30 d) resulted in statistically significant increases in the incubation periods (P<0.01) for five experimental arms . However, the magnitudes of these effects were small and all mice developed neurologic dysfunction. Together, our data indicate that quinacrine is largely inefficacious in all prion-inoculated mice observed.We did not observe obvious signs of acute toxicity resulting from quinacrine treatment in any of the experimental arms. Quinacrine treatment did not substantially alter the progression of disease . Short-t0/0[60–90] mice at 75 and 90 dpi by quantitative LC/MS/MS and comparison to known standards . Three mice were analyzed for each measurement. At 75 dpi, the concentration of quinacrine in the brains of FVB and MDR0/0 mice were ∼1 µM and ∼100 µM, respectively. Quinacrine concentrations were also greater in all other tissues of MDR0/0 mice compared to FVB mice. Similar trends were observed in tissues obtained at 90 dpi. In treated MDR0/0 mice, quinacrine was distributed throughout the brain and was readily detected in the hippocampus, cortex, thalamus, cerebellum, and brainstem .We assessed the accumulation of quinacrine in the brains, kidneys, livers and spleens of FVB[60–90] and MDRtandards Fig. 10/0[60–90] mice and untreated controls at 75 and 90 dpi . The brains of infected MDR0/0 mice showed mild, focal astrocytic gliosis in the thalamus and hippocampus before quinacrine was administered, which was also observed with 15 d of quinacrine treatment (75 dpi). In comparison, the brains of untreated mice at 75 dpi showed moderate to severe astrocytic gliosis in the thalamus and hippocampus, and mild astrocytic gliosis in the cortex. At 90 dpi, astrocytic gliosis in the brains of treated and untreated mice showed subtle differences. At the endpoint of disease, astrocytic gliosis and spongiform degeneration were similar for both treated and untreated MDR0/0 mice. Together, these data suggest that quinacrine has a slight effect on neuropathologic changes upon initial administration, but does not halt widespread neurodegeneration during the course of disease.In parallel, we examined neuropathologic changes in prion-infected MDRd 90 dpi Fig. 2Sc accumulation during the incubation period of MDR0/0[60–90], MDR0/0[0>], and untreated mice. Two or three animals were sacrificed at 60, 75, 90 and 111 dpi, and the accumulation of protease-resistant PrPSc in the brain was analyzed by Western immunoblotting  and quantitative ELISA  following proteinase K (PK) digestion. Additionally, PrPSc levels were measured in MDR[60–90] mice at 7 other time-points . At 60 dpi, MDR0/0[0>] mice had lower PrPSc levels compared to untreated controls. At 75 dpi, PrPSc levels were lower in both MDR0/0[0>] and MDR0/0[60–90] mice compared to untreated controls. However, this difference was not evident at later time-points even with continuous quinacrine administration (MDR[0>] mice). In the absence of quinacrine treatment, PrPSc was readily detectable at 60 dpi. With quinacrine treatment, PrPSc signals were faint but detectable beginning at 75 dpi, a delay of 15 d compared with no treatment. In MDR0/0[60–90] mice, PrPSc was abundant before treatment was initiated (60 dpi), but barely detectable at 69 dpi, indicating that 9 d of quinacrine treatment reduced PrPSc in the brain. These data indicate that quinacrine can both delay the formation and induce the clearance of PrPSc. However, in both cases, PrPSc eventually accumulated in the brains of quinacrine-treated animals until they showed neurologic signs of illness. The transient reduction of PrPSc was not observed in FVB[60–90] mice (data not shown), indicating that accumulation of quinacrine to high levels in the brain is required for prion reduction.We analyzed the kinetics of PrPblotting  and quanSc in the brains of infected mice, we asked if whether quinacrine treatment altered the biochemical characteristics of PrPSc. We prepared 10% brain homogenates of inoculated, untreated MDR and chronically treated MDR[0>] mice at the endpoint of disease. We exposed the samples to increasing concentrations of guanidinium hydrochloride (GdnHCl) for 1 h followed by PK digestion, then quantified the levels of protease-resistant PrPSc by ELISA . As an additional control, we also analyzed untreated brains spiked with 100 µM of quinacrine. PrPSc in the brains of quinacrine-treated mice was slightly less stable than in the brains of untreated controls. With 1.3 M GdnHCl, a significantly greater fraction of quinacrine-exposed PrPSc was denatured compared to quinacrine-naive PrPSc . We also analyzed PrPSc in these brain homogenates using another method, the conformation-dependent immunoassay (CDI) Sc conformations to bind anti-PrP antibodies by measuring the bound ratio between native and denatured samples following partial purification by phosphotungstate (PTA). The CDI measurement for PrPSc purified from quinacrine-treated brains was substantially different from untreatedbrains spiked with quinacrine . Analyses of PrPSc in 10% brain homogenates of these ill mice, using GdnHCl denaturation and CDI as described above, showed no significant differences in the stabilities and CDI measurements , suggesting that the physically altered, quinacrine-induced PrPSc is not a stably propagating strain.We next determined whether the physically altered, quinacrine-treated PrPSc could be formed in cell culture. The re-appearance of drug-resistant PrPSc upon continuous addition of quinacrine to prion-infected N2a cell lines (ScN2a) has not been previously observed. Past experiments were conducted in continuously dividing cultures. Here, we examined the kinetics of PrPSc clearance in infected cells after differentiation by exposure to sodium butyrate or dibutyryl cAMP  to exclude the effect of cell division on prion levels Sc was measured by ELISA.We next asked if quinacrine-resistant PrPryl cAMP Fig. 4Sc and suppressed its accumulation for at least 7 d . In sodium butyrate–treated cultures, PrPSc levels were transiently reduced by exposure to quinacrine, but increased after 2 d of treatment . The observed PrPSc re-accumulation in ScN2a-cl3 cells is similar to that observed in treated MDR0/0 mice and suggests that PrPSc becomes resistant to the effects of quinacrine upon continuous treatment. Using similar treatment, drug resistance does not develop for an alternative antiprion compound, the polyamidoamine (PAMAM) G4 . Quinacrine proved similarly ineffective when infected cells were division-arrested by dibutyryl cAMP . This effect of dibutyryl cAMP is not dependent on activation of the PKA pathway, as the addition of forskolin does not result in a rebound in PrPSc levels . In differentiated cells exposed to sodium butyrate for 4 d prior to quinacrine administration, PrPSc levels only decreased after exposure to quinacrine, indicating that observed changes resulted from quinacrine and not from sodium butyrate exposure . PrPSc slowly increased in untreated, differentiated cells to levels similar to an untreated confluent culture . Unlike the in vivo experiments, the PrPSc that accumulated in division-arrested cells after the addition of quinacrine did not have a significantly altered conformational stability (data not shown). Thus, PrPSc can become quinacrine-resistant without a change in conformational stability as detected by melting of the protein structure with GdnHCl.In dividing ScN2a-cl3 cultures, quinacrine rapidly cleared PrPeast 7 d . In sodieast 7 d . In sodieast 7 d . In sodieast 7 d . In sodieast 7 d . In sodieast 7 d . In sodiSc, we tried to infect N2a cells with lysates from treated cells (data not shown) but were unable to achieve infection. However, lysates from control untreated, infected cells were able to infect N2a cells. We also were unsuccessful at infecting N2a cells with homogenate from the brains of quinacrine-treated mice (data not shown). The difference in infectivity between treated and untreated samples might indicate a conformational change in PrPSc induced by quinacrine. However, given that infection of cell lines with prions is a stochastic phenomenon, we cannot rule out the possibility that the difference in infectivity was caused by chance.Upon addition of sodium butyrate and dibutyryl cAMP, N2a cells became differentiated and could not be further passaged. Instead, in an attempt to propagate quinacrine-treated PrPin vivo. As a minimum requirement for in vivo efficacy, quinacrine must accumulate in the brain at concentrations that exceed its in vitro effective concentration. In chronically treated wild-type mice, the highest nontoxic tolerable dose of quinacrine is 40 mg/kg/day . At this dose, quinacrine accumulates in the brains of FVB mice at a concentration of ∼1 µM. In continuously dividing, ScN2a cells, the EC50 of quinacrine is also ∼1 µM 50 of quinacrine at its site of action may be much greater than its reported EC50 value of 1 µM. We therefore reasoned that the efficacy of quinacrine in vivo might be improved by increasing its steady-state accumulation in the brain to levels approaching its intracellular effective concentration. Because the efflux of quinacrine from the brain is governed by Pgp, an mdr-associated protein mdr1a and mdr1b genes (MDR0/0) or the co-administration of Pgp inhibitors 0/0 mice and achieved drug levels in the brain that were nearly two orders of magnitude higher than those in FVB mice. This level of accumulation far exceeds the intracellular EC50 of quinacrine in culture. Despite its excess accumulation, quinacrine again failed to extend the survival time of prion-inoculated mice. The results indicate that quinacrine's lack of efficacy in vivo is not solely due to its pharmacokinetic properties and may represent a pharmacodynamic failure. The data are consistent with a previous study reporting that continuous intraventricular administration into the cerebral ventricle does not improve the efficacy of quinacrine Quinacrine is a potent antiprion drug in culture but has failed to slow the course of disease in CJD patients and experimental mouse models of prion disease. It is unclear whether this translational gap is due primarily to quinacrine's pharmacokinetic or pharmacodynamic properties 0/0 and wild-type mice. These results suggest that Pgp alone does not significantly influence the accumulation of PrPSc.A recent study reported a decrease in the expression of cerebovascular Pgp in a CJD patient, suggesting a possible role for Pgp in prion pathogenesis Sc levels following drug administration provided insight into the basis of quinacrine's failure in vivo. In the brains of prion-inoculated MDR0/0 mice, PrPSc levels diminished upon the initiation of quinacrine treatment, remained low for several days, but gradually increased despite the continuous presence of the drug. There are two potential explanations for this trend: (1) target tissues, such as the brain, alter their responsiveness to quinacrine or (2) prions become resistant to the actions of the drug. There is precedence for the former phenomenon. It has been shown that during chronic administration, various drugs and steroid hormones lose efficacy by inducing the expression of efflux transformers and cytochrome P450 Sc conformations that survived the initial treatment. A similar linkage between limited efficacy and the formation of drug-resistant prions was cited for amyloidophilic compounds Sc accumulating in the brains of chronically treated mice had a lower conformational stability compared to that of untreated controls, suggesting that quinacrine induced a structural change within the prion population. Second, we were able to induce the formation of quinacrine-resistant prions in cell culture, indicating that drug resistance can be induced outside the context of the central nervous system.Kinetic analysis of PrPSc in a cell is established by the balance between its rates of formation and clearance. As has been noted before Sc in a dividing cell is the sum of the rate of catabolism and the rate of cell division. Thus, the apparent rate of clearance for a given prion conformation is likely to be slower in stationary cells compared to dividing cells. In a dividing cell, the process of cell division artificially enhances the rate of clearance and prevents the accumulation of PrPSc. Therefore, the probability that a partially resistant conformation survives quinacrine treatment is increased in stationary cells. These observations suggest that conducting drug screens in stationary cells may be more likely to identify antiprion compounds that prove effective in vivo.Interestingly, the formation of quinacrine-resistant prions is only observed in division-arrested cultured cells. The steady-state concentration of PrPSc conformations Sc rebound observed in this study is reminiscent of the failure of antiviral drugs caused by selection of drug-resistant variants. However, unlike resistant viral strains, the quinacrine-resistant conformation formed here was not able to propagate in the absence of the drug and thus cannot be considered a stably propagating strain. In the absence of quinacrine, the initial physical properties of the prion population are rapidly re-established. In viral therapy, the administration of drug cocktails, combining compounds with differing modes of action, reduces the probability of virologic failure. Ultimately, the co-administration of multiple antiprion compounds may be required in order to avoid the formation of resistant prion conformations. Additionally, targeting PrPC, the endogenous substrate shared by all prion strains, is likely to identify compounds that have broad specificity and are uniformly effective against all strains.Increasing evidence shows that prion-infected tissues harbor multiple, distinct PrPQuinacrine dihydrochloride, PAMAM G4, forskolin and sodium butyrate were purchased from Sigma-Aldrich . N2a cells were obtained from American Tissue Culture Collection. Minimal essential medium (MEM) with Earle's salts; Dulbecco's Modified Eagle Medium (DMEM) High Glucose 1× with 4.5 g/L D-glucose and L-glutamine and without sodium pyruvate; cell dissociation buffer; fetal bovine serum (FBS); Geneticin (50 mg/mL); penicillin-streptomycin ; and GlutaMAX were purchased from Gibco. Dithiothreitol ; 4× loading buffer; and proteinase K (PK) were purchased from Invitrogen. Complete protease inhibitor (PI) cocktail tablets were from Roche Diagnostics. Western blotting detection reagents 1 and 2 were from GE Healthcare. Anti-PrP antibodies Fab D18 0/0 mice were purchased from Charles River  and Taconic , respectively. Mice were inoculated intracerebrally with 30 µL of 1% brain homogenate containing the RML prion strain. Treatment consisted of 40 mg/kg/day of quinacrine in a chocolate-flavored liquid diet 0/0 mice; 60 dpi for FVB mice; and 70, 80, 95, and 105 dpi for CD1 mice. The initiation of quinacrine treatment varied for each mouse strain to reflect the relative incubation period of the respective mouse line.All protocols were approved by the University of California San Francisco Animal Care and Use Committee. Approximately five-week-old male and female wild-type (FVB and CD1) and MDRAt 75 and 90 dpi, 2–3 mice were euthanized. The left half-brain was snap frozen on powdered dry ice for quinacrine extraction and biochemical analysis; the right half-brain was fixed for pathological analysis. Brain homogenates (10% w/v) were prepared using a Precellys 24 homogenizer  in pure distilled water  and were aliquoted and stored at −80°C.Animals were observed every day for signs of neurologic disease. Mice were diagnosed with prion disease when they exhibited three or more of the following neurological symptoms: ataxia, circling, depression, and blank stare. Upon diagnosis, mice were killed and their brains collected as described above for analysis.g for 5 min. LC/MS/MS system consisted of Shimadzu LC-10 AD pumps, a Waters Intelligent Sample Processor 717 Plus autosampler, and a Micromass Quattro LC Ultima triple quadruple tandem mass spectrometer. The mass spectrometer was set to electrospray ionization in the positive-ion mode. Quinacrine and its metabolites, O-demethylated quinacrine (M1) and mono-desethyl quinacrine (M2), were monitored by multiple-reaction monitoring (MRM) at 400.5>142.2 m/z for QA, 384.5>142.2 m/z for M1, 372.2>114.2 m/z for M2, and 277.2>142.2 m/z for internal standard (chlorphenamine). The column was a Betasil C18 column (50×4.6 mm) from Hypersil-Keystone and the mobile phase consisted of CH3OH/H2O/trifluoroacetic acid (45∶55∶0.05) with 1 mM ammonium formate. The flow rate was 0.8 mL/min.Quinacrine was extracted from brain samples and its concentration was measured by LC/MS/MS as described previously Ten percent brain homogenates (100 µL) were diluted with 1 mL lysis buffer and digested with 20 µl of 1 mg/mL PK at 37°C for 1 h. PMSF  was added to stop the digestion. Sample volumes of 200 µl were used for ELISA analysis and of 800 µl for Western blot analysis.ELISA plates were prepared as described previously Sc pellets from 800 µl of PK-digested samples were collected and resuspended with lysis buffer. SDS sample running buffer and reducing reagent (Invitrogen) were added. Samples were heated to 95°C for 5 min and run in a 4–12% Tris-glycine SDS gel (Invitrogen). The gel was transferred to PVDF membrane using an iBlot (Invitrogen) and the membrane was blocked with 5% milk for 30 min. The membranes were subsequently incubated overnight with D13-HRP antibody and washed 3 times with TBST for 5 min before developing with ECL reagent.For Western blot analysis, PrPBrain homogenates (10 µL) were incubated with GdnHCl, in a range of 0 to 2 M, at 22°C for 2 h. The samples were subsequently diluted with lysis buffer to a final concentration of 0.4 M of GdnHCl. PK was added at a final concentration of 50 µg/mL and the samples were incubated at 37°C for 1 h. PMSF  was added to stop PK digestion. The samples were subsequently analyzed by ELISA as described above.3. The plates were washed 3× with TBS, pH 7.8, containing 0.05% (v/v) of Tween 20, then incubated at room temperature for 2 h. HRP-labeled D13 anti-PrP antibody was added and incubated as described Each sample was divided into two aliquots, precipitated by the addition of 1% PTA and resuspended in lysis buffer. The first aliquot was untreated and designated native; the second aliquot was mixed to a final concentration of 4 M GdnHCI, heated for 20 min at 80°C, and designated denatured. Both samples were diluted 20-fold with lysis buffer containing protease inhibitors . Then, 200 µl volumes were loaded on 96-well polystyrene plates that were previously coated with the mAb D18. The plates were blocked overnight at 18–22°C in 0.2 M phosphate buffer, pH 7.2, and then blocked with TBST, pH 7.2, containing 0.25% BSA (w/v) and 6% Sorbitol (w/v), and 0.03% (w/v) NaN2O2 in methanol for 20 min. The slides were washed 3 times for 5 min using PBS with 0.1% Tween 20 (PBST) with 3 buffer changes. The glial fibrillary acidic protein (GFAP) target antigen does not require retrieval for antibody recognition . Nonspecific antibody binding was blocked with 5% normal goat serum (NGS) in PBST for 30 min, then the slides were incubated with the primary antibody at 1∶1000 in PBST at 4°C overnight. After washing, the sections were incubated with a biotinylated goat anti-rabbit secondary antibody (Pierce) at 1∶1000 in PBST with 5% NGS for 1 h at room temperature. After additional washing, the Vector ABC kit was used per the manufacturer's instructions. Sections were washed and developed with the Vector DAB kit for 3 min and washed again. Tween 20 was not present in the third wash. The sections were counterstained for 10 s in hematoxylin (Fisher), taken through graded alcohols to xylene, and coverslipped using Permount (Fisher). Images were taken at 20× and 40× magnifications using the SpotFlex camera and program on the Leica DM-IRB microscope.After mice were euthanized, their right half-brains were fixed in 10% formalin for a minimum of 3 d. Fixed brains were processed and embedded in paraffin, and 8-µm sections were cut from four representative brain regions: cortex, cerebellum, hippocampus and thalamus. Slides were deparaffinized and endogenous peroxidases blocked with 3% HThe PrP-overexpressing N2a-cl3 cell line was created by stably transfecting N2a cells with the pSPOX.neo vector expressing full-length mouse PrP under the control of the hCMV promoter with DOTAP liposomal transfection reagent (Roche). Stably transfected lines were cloned by serial dilution and 15 individual clones were assayed for PrP expression level by Western immunoblotting. The N2a-cl3 clone overexpresses PrP at ∼6× the levels of untransfected N2a cells. N2a-cl3 cells were infected with RML prions as previously described


We compared the ability of two devices estimating cardiac output from arterial pressure-curve analysis to track the changes in cardiac output measured with transpulmonary thermodilution induced by volume expansion and norepinephrine in sepsis patients.In 80 patients with septic circulatory failure, we administered volume expansion (40 patients) or introduced/increased norepinephrine (40 patients). We measured the pulse contour-derived cardiac index (CI) provided by the PiCCO device (CIpc), the arterial pressure waveform-derived CI provided by the Vigileo device (CIpw), and the transpulmonary thermodilution CI (CItd) before and after therapeutic interventions.2) as well as by norepinephrine . The changes in CIpc accurately detected an increase in CItd ≥ 15% induced by volume expansion and norepinephrine introduction/increase  and 0.924 (0.795 to 0.983), respectively; P < 0.05 versus 0.500 for both). The changes in CIpw were less reliable for tracking the volume-induced changes in CItd  and norepinephrine-induced changes in CItd . The changes in CIpw were unable to detect an increase in CItd ≥ 15% induced by volume expansion and norepinephrine introduction/increase  and 0.541 .The changes in CIpc accurately tracked the changes in CItd induced by volume expansion (bias, -0.20 ± 0.63 L/min/mThe CIpc was reliable and accurate for assessing the CI changes induced by volume expansion and norepinephrine. By contrast, the CIpw poorly tracked the trends in CI induced by those therapeutic interventions. Cardiac output is regarded as one of the most important variables to be monitored in patients with acute circulatory failure . PiCCO aNevertheless, these two techniques differ in the way they estimate aortic impedance. The PiCCO device provides an estimation of the cardiac index from an analysis of the pulse contour (CIpc), but it does not estimate aortic impedance because it calibrates CIpc from a measurement of cardiac index obtained from transpulmonary thermodilution (CItd), a technique that has demonstrated a robust accuracy in comparison with pulmonary artery thermodilution -9. In coTherefore, we assessed the respective abilities of CIpc and CIpw to track the changes in CItd induced by volume expansion or by norepinephrine in sepsis patients.After approval by our Institutional Review Board (Comité pour la Protection des Personnes île-de-France VII), we enrolled 80 patients who had circulatory failure of septic origin in our medical intensive care unit. Acute circulatory failure was defined by the presence of one or more of the following signs: (i) systolic arterial pressure ≤90 mm Hg ; (ii) urinary flow ≤0.5 ml/kg/hr for more than 2 hours; (iii) tachycardia ≥100 beats/min; and (iv) skin mottling ,26. PatiIn 40 of these patients (Group 1), the attending physician decided to administer fluid because the patient showed some criteria predicting fluid responsiveness: increase in cardiac index (CI) ≥10% during a passive leg-raising test ; increasAll patients had a catheter inserted into the internal jugular vein and a catheter inserted into the femoral artery . The arterial line was divided in two branches, one connected to a PiCCOplus device , and the other one connected to a FloTrac/Vigileo device . This enabled the two devices simultaneously to analyze the same sample of the arterial pressure curve.Before each therapeutic intervention , we performed a first set of hemodynamic measurements, including heart rate, systemic arterial pressure, CItd, CIpc, CIpw, and systemic vascular resistance (SVR). We used the values of CIpc and CIpw that were automatically displayed on the screens of the commercial devices. The CIpc and CIpw displayed by the monitors are averaged over a 12-second and 20-second rolling period, respectively. We could not obtain the digital  data from the devices. In practice, the CIpc and CIpw values displayed by the monitors were averaged over 10-second periods. The CItd was measured by the PiCCOPlus device by injecting 15 ml of iced saline (<8°C) through the central venous line. The injection was manually performed in triplicate, and the values of CItd were averaged. Immediately after performing thermodilution boluses, the values of CIpc were measured, the arterial line being physically shared in Y through taps toward the Vigileo and the PiCCO devices. Because the thermodilution automatically calibrated the pulse-contour analysis of the PiCCO device, CIpc was identical to CItd at the starting time. The CIpw was carried immediately before thermodilution to avoid interference between the temperature drift and the accuracy of the CIpw. The total SVR was estimated as mean arterial pressure divided by CItd.After the first set of hemodynamic measurements was completed in Group 1, fluid loading was performed by infusing 500 ml of saline over a 30-minute period. In Group 2, norepinephrine was titrated, targeting a mean arterial pressure of 65 mm Hg . In each patient, we performed only one pair of measurements (before/after therapeutic intervention). Comparisons between values recorded before with values recorded after therapeutic interventions were performed in both groups by using a paired Student t test or a paired Wilcoxon test, as appropriate. Comparisons between Group 1 and Group 2 were performed with a two-tailed Student t test or a Mann-Whitney U test, as appropriate.All data were normally distributed (Kolmogorov-Smirnov test), except CItd, CIpc, CIpw, and the dosage of norepinephrine, and are expressed as median [25We compared the relative changes of CIpc and of CIpw with those of CItd during the therapeutic interventions by a Bland and Altman analysis (for absolute changes) and by linear regression analysis (for percentage changes). We tested the ability of CIpc and CIpw to detect an increase in CItd ≥ 15% by constructing receiving operating characteristics (ROC) curves. The area under the ROC curves  were compared by using the Hanley-MacNeil test. This analysis was also separately performed in patients in whom the SVR changed (increased or decreased) by >15% with the therapeutic interventions and in patients in whom the SVR changed (increased or decreased) by <15% with the therapeutic interventions . The valThe precision of each method was calculated from data obtained from a sample made of the first 20 and the last 20 patients included in the study when arterial pressure was stable. For CItd, we calculated the coefficient of variation (ratio of the standard deviation to the mean) for each set of three consecutive thermodilution boluses and then averaged it for the series of the 20 sets. For CIpc and CIpw, we collected the 10 consecutive values of CI displayed on the monitor. We calculated the coefficient of variation for each set and averaged it for the series of 20 sets .P value < 0.05 was considered significant. The statistical analysis was performed by using Statview 5.0 software .A The characteristics of the patients at baseline are summarized in Table Considering the population as a whole, the therapeutic interventions significantly increased the mean arterial pressure, CItd, CIpc, CIpw, and the SVR by 21 (5 to 38)%, 13 (6 to 22)%, 14 (4 to 20)%, 8 (0 to 18%), and 5 (-10 to 19)%, respectively.2. The coefficient of correlation between the percentage changes induced by the therapeutic interventions in CIpc and in CItd was 0.73 (P < 0.05). After the therapeutic intervention, the bias between the absolute values of CIpc and CItd was -0.07 ± 0.36 L/min/m2, and the percentage error was 22%.The bias between the absolute changes in CIpc and CItd induced by therapeutic interventions was -0.07 ± 0.73 L/min/m2. The coefficient of correlation between the percentage changes induced by the therapeutic interventions in CIpw and in CItd was 0.006 (P = 0.11). After the therapeutic intervention, the bias between the absolute values of CIpw and CItd was -0.09 ± 0.94 L/min/m2, and the percentage error was 61%.The bias between the absolute changes in CIpw and CItd induced by therapeutic interventions was -0.10 ± 1.40 L/min/mIn Group 1, the volume expansion significantly increased the mean arterial pressure, CItd, CIpc, and CIpw by 9 1 to 20)%, 14 (7 to 24)%, 10 (4 to 16)%, and 7 (0 to 18)%, respectively. The SVR nonsignificantly decreased by -10 [0 to -16)%   Table . An incr2. The coefficient of correlation between the fluid-induced percentage changes in CIpw and in CItd was 0.33 (P < 0.05)  Table . An incrIn Group 2, the introduction or increase of norepinephrine significantly increased the mean arterial pressure, CItd, CIpc, CIpw, and the SVR by 21 (5 to 36)%, 13 (7 to 22)%, 10 (3 to 17)%, 8 (0 to 18)% and 19 (9 to 31)%, respectively (Table 2. The coefficient of correlation between the norepinephrine-induced percentage changes in CIpc and in CItd was 0.78 (P < 0.05)  Table . An incr2. The coefficient of correlation between the norepinephrine-induced percentage changes in CIpw and in CItd was -0.003 (P = 0.65) .Considering the whole population, the SVR increased by 5 (-10 to 19)%. The bias between the changes in CIpc and CItd was not significantly correlated with the changes in SVR. By contrast, the bias between the changes in CIpc and CItd was significantly correlated with the changes in SVR , and the coefficient of correlation between the percentage changes in CItd and CIpw was 0.47 (P < 0.05).In the subset of patients in whom the SVR changed by <15% (n = 36), the bias between the absolute changes in CIpc and CItd was -0.07 ± 0.63 L/min/m2, and the bias between the absolute changes in CIpw and CItd was -0.06 ± 1.79 L/min/m2. The coefficient of correlation between the percentage changes in CItd and CIpc was 0.78 (P < 0.05), and the coefficient of correlation between the percentage changes in CItd and CIpw was -0.15 (P < 0.05).In the subset of patients in whom the SVR increased by >15% (n = 44), the bias between the absolute changes in CIpc and CItd was -0.08 ± 0.70 L/min/m2; bias, -0.08 ± 0.40 L/min/m2). In these patients after the therapeutic intervention, the bias between the absolute values of CIpc and CItd was -0.11 ± 0.22 L/min/m2, and the percentage error was 14%. After the therapeutic intervention, the bias between the absolute values of CIpw connected to the radial line and CItd was -0.47 ± 0.84 L/min/m2, and the percentage error was 57%.Considering the 20 patients in whom the Vigileo was successively connected to the radial and the femoral arterial lines before and after the therapeutic intervention (40 pairs of measurements), the CIpw values measured from the radial and the femoral lines were not statistically different (2.9 (2.6 to 3.1) vs. 2.8 (2.6 to 3.1) L/min/m2; bias, 0.02 ± 0.31 L/min/m2). The CIpw was not different when the femoral arterial line was connected only to the PiCCO device or when it was directed in a Y to the PiCCO and the Vigileo devices (2.9 (2.6 to 3.2) vs. 2.9 (2.6 to 3.2) L/min/m2; bias, 0.04 ± 0.38 L/min/m2).Considering the 20 patients in whom the femoral arterial line was alternatively directed toward the sole PiCCO or Vigileo devices or directed in a Y to both, before and after therapeutic interventions (40 pairs of measurements), the CIpc was not different when the femoral arterial line was connected only to the PiCCO device or when it was directed in a Y to the PiCCO and the Vigileo devices (3.1 (2.6 to 3.8) vs. 3.0 (2.8 to 3.9) L/min/mThe coefficient of variation was 6.8% for the CItd, 1.8% for CIpc, and 2.0% for CIpw.We have shown that the calibrated pulse contour-derived CI accurately tracked the changes in CI induced by volume expansion and norepinephrine in sepsis patients. By contrast, the uncalibrated arterial pressure waveform-based CI tracked the changes in CI induced by those therapeutic interventions with less accuracy. The more the SVR changed, the less the uncalibrated arterial-pressure waveform analysis was accurate for monitoring the changes in CI.Among the techniques that have been developed as an alternative to the pulmonary artery catheter, transpulmonary thermodilution has been demonstrated to be reliable for measuring cardiac output as compared with classic thermodilution -6,8,9 anDifferent commercial devices use the arterial-pressure waveform analysis for providing a real-time estimation of stroke volume and cardiac output. The PiCCO device calculates stroke volume by measuring the area under the systolic portion of the arterial-pressure curve and dividing it by the aortic impedance. The latter is determined by calibration against a measure of cardiac output by transpulmonary thermodilution. In addition, this device enables tracking the changes in arterial compliance. At the time of calibration, arterial compliance is calculated from the time constant of the pressure decay in diastole (t) and SVR (compliance t/SVR). Then, compliance and resistance are updated from beat to beat, according to a proprietary algorithm that depends particularly on the arterial pressure (P) and on dP/dt. A specific patient-calibration factor  (which is independent of compliance and resistance) is added to the formula, which computes the pulse-contour cardiac output . This esThe Vigileo device differs from the PiCCO device in two main points. First, it does not take into account the area under the systolic part of the arterial curve, but does the standard deviation of the points contained by the arterial curve in a beat. Second, it does not determine aortic impedance from any external calibration of cardiac output, but estimates it from pressure-waveform characteristics, such as skewness and kurtosis, and from patient demographic data  . With thIn the present study, the CIpc showed good accuracy in tracking the CItd changes induced by the therapeutic interventions. Moreover, it was able to detect correctly the increases in CItd of ≥15% in ROC curve analysis. Interestingly, this ability was not altered by the level of the changes in SVR, confirming what we recently reported . It is nBy contrast, in our sepsis patients, the uncalibrated estimation of CI poorly tracked the short-term changes in CItd during volume expansion and in those secondary to norepinephrine introduction/increase. Furthermore, the CIpw was unable detect correctly the changes in CItd ≥ 15%, induced either by volume expansion or by norepinephrine introduction/increase. The precision of CIpw device was high  , but itsFirst, the CIpc was calibrated at baseline such that before therapeutic interventions, the CIpc was similar to the CItd. In this regard, it could be considered that the CIpc was advantaged compared with the CIpw. Nevertheless, the calibration of the CIpc was performed only at baseline and thus could not affect the comparison of the ability of CIpc and CIpw to track changes in CI induced by the therapies.Second, we did not use the pulmonary artery thermodilution as a reference. Nevertheless, the accuracy of the transpulmonary thermodilution to measure cardiac output has been repeatedly demonstrated ,32,34-37Third, we investigated the reliability of CIpc and CIpw to track short-term changes in CI. If tested during a longer calibration-free period , the reliability of CIpc could be less. Finally, we used the data that were automatically displayed by both devices . Thus, we could not precisely analyze the reason that one device performed differently. The filtering and averaging of data by the devices also likely explained part of the low variability of CIpc and CIpw.The pulse contour-derived estimation of CI provided was accurate for assessing the changes in CI induced by volume expansion and norepinephrine in sepsis patients. By contrast, the uncalibrated estimation of CI by arterial-pressure waveform analysis was not of sufficient accuracy for monitoring CI changes in this setting. Whether a new generation of the Vigileo device would perform better than the current commercial model we used remains to be demonstrated.• In septic shock patients, the pulse contour-derived estimation of cardiac index correctly tracked the changes in cardiac index induced by volume expansion and norepinephrine.• The pulse-wave analysis was less reliable for tracking the changes in cardiac index induced by those therapeutic interventions.• The ability of the pulse-wave analysis to track the changes in cardiac index was poorer when the systemic vascular resistance changed to a large extent.• Whether more-recent versions of the devices could perform differently should be assessed in further studies.CI: cardiac index; CItd: CI measured by transpulmonary thermodilution; CIpc: CI measured by the PiCCO pulse-contour analysis; CIpw: CI measured by FloTrac/Vigileo pressure-waveform analysis; SVR: systemic vascular resistance.Professors Jean-Louis Teboul and Xavier Monnet are members of the Medical Advisory Board of Pulsion Medical Systems. The other authors have no financial competing interest to disclose.XM conceived the study, contributed to the collection of data, performed analysis and interpretation of the data, and drafted the manuscript. NA contributed to the collection of data. BN contributed to the collection of data. JJ contributed to the collection of data. CR participated in the coordination of the study and helped to draft the manuscript. J-LT conceived the study, coordinated the collection, analysis, and interpretation of the data, and helped to draft the manuscript. All authors read and approved the final manuscript.


Androgen insensitivity syndrome (AIS) is a rare disease associated with inactivating mutations of AR that disrupt male sexual differentiation, and cause a spectrum of phenotypic abnormalities having as a common denominator loss of reproductive viability. No established treatment exists for these conditions, however there are sporadic reports of patients (or recapitulated mutations in cell lines) that respond to administration of supraphysiologic doses (or pulses) of testosterone or synthetic ligands. Here, we utilize a novel high content analysis (HCA) approach to study AR function at the single cell level in genital skin fibroblasts (GSF). We discuss in detail findings in GSF from three historical patients with AIS, which include identification of novel mechanisms of AR malfunction, and the potential ability to utilize HCA for personalized treatment of patients affected by this condition. While in the cytoplasm under baseline conditions, upon addition of agonist AR translocates to the nucleus, where it interacts with coregulators and promoters/enhancers of AR-responsive genes, and regulates transcription. AR action is a 3H-DHT dissociation constant (Kd) and maximal binding (Bmax), but increased ligand-receptor dissociation rate can be normalized under certain culture conditions. For instance, such an AR containing a single amino acid substitution (Y763C) and a reduced polyglutamine tract (Q12) normalized its transcriptional activity when exposed to pharmacologic concentrations of androgens both in vitro Despite the clinical dogma that “AIS is not treatable,” some sporadic PAIS and MAIS patients respond to endocrine management consisting of pharmacologic doses of androgens Our group has recently developed a high throughput microscopy-based technology to simultaneously analyze multiple AR activities at the single cell level, an approach often referred to as high content analysis (HCA). Our AR-oriented HCA involves multi-parametric interrogation of cultured cells using automated high magnification, high resolution imaging and immunofluorescence or green fluorescent protein-fused AR (GFP-AR) in combination with use of a red fluorescent protein-based transcriptional reporter protein d, 2) normal Bmax, and 3) qualitative abnormalities of ligand-receptor interaction would be the most amenable to normalization. We present here unique cytological profiles generated by HCA from three historical patients affected by CAIS or PAIS, which were complemented by NH2-COOH-terminal domain interaction (NC-TDI) experiments, and previously published ligand-binding studies We theorized that HCA of AR would be an ideal technology to directly analyze patient-derived genital skin fibroblasts to identify not only mechanisms associated with abnormal AR activities, but also therapeutic options normalizing mutant AR functions. While there are many AIS mutations described, those localized in the LBD and associated with 1) normal KPrimary cultures of genital skin fibroblast from AIS patients F764L, R840C and P766S, and six normal patients were established as previously described http://androgendb.mcgill.ca/). The discrepancies are due to the different number of glutamines in the polyglutamine region of exon 1 that were reported in the initial sequences of AR.The mutations investigated in this study are AR-F764L, AR-R840C, and AR-P766S. These mutations were previously identified in patients affected by either PAIS, or CAIS, and functionally characterized by using either patient derived genital skin fibroblasts, or after transfection in CV1 or CHO cells 3H-DHT Kd and Bmax , and abnormally elevated ligand dissociation rate The F764L mutation was originally identified in a CAIS patient of Dr. G. Costin  3H-DHT Bmax and Kd and presence of thermolability This mutation has been identified in multiple patients with PAIS d and Bmax, and abnormally elevated ligand dissociation rate. Transcriptional activity was 9% of control in response to DHT, but normalized at 120% of control in response to Mibolerone This mutation was identified in three patients with CAIS 3H-DHT Kd and Bmax. According to the series of 130 families published by Dr. Griffin, these features are shared by 40% of patients with AIS This historical analysis showed that AR-R840C, AR-F764L and AR-P766S shared these following common features; they were point-mutations localized in the ligand binding domain with normal 2-terminal GFP tag and regulation by the CMV promoter has been previously described cDNA expression vectors for hAR harboring the AIS related mutations were previously generated A HeLa cell line stably expressing wild type GFP-AR was previously described 2 terminal sequence F23XXLF27 is an important step toward the activation of AR 6 Hela cells were seeded into a 60 mm dish. Cells were co-transfected with 1.3 ug of pFN11A-AR624-919, 1.3 ug of pFN10A-1-660, 1.3 ug of pGL4.31 , and 1 ng of phRL-TK carrying the Renilla luciferase gene. After 12 hours of transfection, cells were trypsinized and equally seeded into nine wells of a 12 well plate, with one well treated with vehicle and others with various concentration of different ligand. After 24 h of treatment, luciferase activity was assayed with the Promega Dual Glo assay kit, using a luminometer (PerkinElmer). Data represent the average±SD of three independent experiments, are expressed as fold-induction compared with negative control, and represent the units of firefly luciferase corrected for the units of renilla luciferase detected in the same plate.An interaction between COOH terminal AF2 (activation function 2) and NH2PB-dsRED2skl reporter plasmid, consisting of the AR responsive promoter ARR2PB To determine the global effect of the various AIS mutations on AR signaling, each was analyzed using HCA. This technology allows simultaneous quantification of AR protein expression, nuclear translocation, subnuclear patterning, and transcriptional activity 2PB-dsRED2skl, cells were plated onto 100 mm plastic dishes in medium supplemented with 5% charcoal-stripped and dialyzed-FBS. Transient introduction of the pARR-2PB-dsRED2skl reporter construct was performed using 6.0 µg reporter plasmid and 6.0 µg carrier DNA  using Transfectin (Biorad) following standard protocols. After 8-hour incubation, DNA/lipid complexes were removed. Cells were then trypsinized and replated at 10,000 cells per well in Nunc poly-D-lysine treated 96-well optical glass bottom plates and incubated an additional 12 hours to allow for cell adhesion. Cells were then exposed to DHT, R1881 or Mibolerone for 24 h. Compound dilutions and final addition to multi-well plates were performed using a Beckman Biomek NX robotic platform to ensure repeatability from experiment to experiment. After incubation was complete, using the Biomek NX robot, plates were washed with PBS and fixed for 20 min at RT in 4% formaldehyde prepared in CSK buffer . After fixation, cells were briefly permeabilized (5 min) with 0.5% Triton-X and prepared for imaging by washing in PBS, aspirating the washed solution, and adding a 1 ng/ml DAPI solution to stain DNA and a generic protein stain  to facilitate cell and nuclear image segmentation. Cells were imaged in PBS.For studies utilizing HeLa GFP-AR cell lines, twenty-four hours before transfection with pARR4Cl solution for 10 minutes. Next, cells were permeabilized (30 min) with 0.5% Triton-X. After washing, cells were incubated for 30 minutes in 5% non-fat milk in Tris buffered Saline (TBS)-Tween (Blotto) followed by an overnight incubation in Blotto containing a 1 µg/ml anti-AR mouse monoclonal antibody  with 4 images captured per field . In general, 49 to 64 images were captured per well for image analysis.Cells were imaged using the Cell Lab IC-100 Image Cytometer  platform that consists of 1) Nikon Eclipse TE2000-U Inverted Microscope ; 2) Chroma 82000 triple band filter set ; 3) an imaging camera: Hamamatsu ORCA-ER Digital CCD camera ; and 4) a focusing camera: Photonics COHU Progressive scan camera . The microscope was equipped with a Nikon S Fluor 40X/0.90NA objective and the imaging camera set to capture 8 bit images at 2×2 binning  DNA content (DAPI sum of pixel intensities) . All other responses with ligand treatment are reported at fold change compared to untreated samples . Determination of significant differences between compounds was accomplished by first performing an ANOVA analysis followed by a post-hoc multiple comparison analysis with significance set at <0.05.Applied gates were based upon 1) nuclear area; 2) nuclear circularity [(4*π*area)/perimeternsities) . AR expr2 values close to 1, regardless of the ligand or AR construct used. An example of this analysis for HeLa cells stably transfected with wild type AR, AR-F764L, AR-R840C or ARP766S and treated with DHT is shown in 2 value ranged between 0.85 and 0.999, and p values were <0.0001.Pearson's correlations analysis was performed to verify the relationship existing between AR nuclear hyperspeckling and transcriptional activation in HeLa cells. For each mutant and ligand used, a strong positive correlation between these two parameters was confirmed, with RAR-F764L did not respond to T and DHT, however it responded partially to R1881 (1.8-fold at 200 nM), and maximally (3.1- fold at 200 nM) to Mibolerone. NC-TDI increased from 10% to 50-60% of wild type receptor at 0.2 and 200 nM of Mibolerone, respectively. Thus, increasing the concentration of Mibolerone to suprasaturating concentrations elicited a partial rescue of NC-TDI in mutant AR- F764L , in the range observed in normal GSFs, at EC50 of 0.14-0.61 nM , Table 3All three agonists induced significant nuclear translocation, hyperspeckling and transcriptional activation of AR-P766S to wild-type AR levels, however, these responses were observed at significantly higher concentrations of ligand , Table 1All three agonists induced significant nuclear translocation (14.3%–20.7%) when used at higher concentrations (EC50: 1.2–3.2 nM) in GSFs harboring the AR-P766S mutation , Table 33H-DHT Bmax and Kd may be reversible, and proved this by using the new AR HCA technology and conventional assays, such as NC-TDI. HCA not only proved to be a powerful tool to understand the functional abnormalities of AR mutations directly in patient-derived specimens, but was also utilized to predict clinical responses to a variety of experimental treatments.We hypothesized that AR point mutations localized in the ligand binding domain and associated with normal 2-COOH terminal interaction, and if so, if this impairment would be amenable to normalization using conditions that enhance stability of binding, for instance by increasing concentrations of natural or synthetic AR agonists. NC-TDI was impaired in the two mutations associated with increased ligand-receptor dissociation rate; AR-F764L and AR-P766S. ARF764L demonstrated a ligand-dependent partial rescue of NC-TDI to approximately 50% of wild type, at high concentrations (e.g.: 200 nM) of Mibolerone, but not of R1881 and DHT. AR-P766S demonstrated a complete rescue of NC-TDI with all four ligands, in a way that was proportional to the concentration used, and reached complete normalization at 200 nM. These data confirmed that notion that abnormal NC-TDI is a hallmark of increased ligand-receptor dissociation rate 3H-DHT binding causing thermolability, not increased ligand-receptor dissociation rate. Thus, it should not have been a surprise to find that NC-TDI was unaffected in AR-R840C. The mechanism of androgen insensitivity in the context of an AR mutation showing increased NC-TDI is unclear. Because AF2 is important not only for NC-TDI, but also for p-160 coactivators recruitment Experiments were performed to establish if the three mutants were affected by an impairment of the NHHCA activities measured in primary GSF cultures from six normal patients that were used as a control were within a relatively small interval. These minor differences probably reflect a unique intracellular equilibrium of molecules regulating AR activation, or polymorphisms of the polyglutamine repeat of AR exon 1. It is also possible that the different HCA activities observed in these cell lines reflect different degrees of normal virilization that are present in the general population.First, as previously described Second, the EC50's for hyperspeckling were lower in GSF compared with HeLa by a factor of approximately 70-fold. Third, the fold-induction of hyperspeckling  was lower by a factor of 5-fold in GSF cells compared to HeLa-AR. The reasons for these discrepancies are not clear. The first discrepancy may be related to decreased ability of ligand-bound AR to interact with the machinery necessary for nuclear import in HeLa cells, or to the presence of a competing androgen-binding protein in the cytosol but not the nucleus of HeLa, so that higher concentrations of androgen would be necessary to complete functions that take place in the cytoplasm, such as nuclear import, but not in the nucleus, such as hyperspeckling or transcriptional activity. The third discrepancy may be related to decreased transcriptional activity in GSF reported by others A number of reproducible, cell line-dependent differences were detected in the control groups between HeLa and GSF. These studies have shown that the recovery of AR-F764 activity (e.g.: 45% of control) occurs in a Mibolerone-dependent way. Similar results were obtained in GSF containing ARF764L or transfected HeLa; the main difference being that Mibolerone was more effective in GSF, with a recovery of hyperspeckling, a surrogate marker of transcription, at 100% of control levels. These studies reconcile the CAIS phenotype of this patient with the fact that at the critical time of genital differentiation the fetus was exposed to the endogenous ligand DHT, which is inactive with AR-F764L. A Mibolerone-specific recovery of function argues that this agonist fits inside the mutated ligand-binding pocket of AR-F764L more properly than the physiologic ligand DHT. Stabilization of the receptor-ligand complex with high doses of Mibolerone rescued NC-TDI to 50% of control, and this was associated by improved ligand retention in the ligand-binding pocket of the receptor, and by an increase in transcription.We observed complete recovery of AR NC-TDI and HCA functions with pharmacologic concentrations of DHT, R1881 and Mibolerone for mutant AR-P766S. Because the interaction between ligands and AR-P766S was highly unstable at physiologic concentrations, NC-TDI and transcription were compromised to the point that the patient developed a CAIS phenotype. Supraphysiologic concentrations of the three ligands improved ligand-receptor stability, and normalized NC-TDI and transcriptional activity to control levels.HCA analysis in HeLa and GSF cells showed that nuclear translocation of AR-R840C could be almost normalized at higher concentration of each ligand, however neither hyperspeckling nor transcriptional activity were recovered. Because this mutation was not associated with instability of receptor-ligand binding, lack of response to experimental conditions coupled with increased receptor-ligand stability was not surprising. As stated above, the presence of increased NC-TDI may be associated with decreased recruitment of coactivators and decreased transcriptional activity.First, AIS mutations with normal Kd, Bmax, and increased ligand dissociation rate can be rescued. In contrast, other mutations with a slightly different phenotype  were not rescued. Thus, to this point the biochemical signature of AR mutations that can be rescued consists in normal Kd, Bmax, and increased ligand dissociation rate. In support of these observations, the PAIS patients who responded to treatment in the paper of Grino Second: mutations with normal Kd and Bmax and increased ligand dissociation rate are associated with decreased NC-TDI. NC-TDI is a surrogate of ligand-receptor binding stability, thus such mutations can be rescued by using conditions that increase ligand-receptor binding stability, such as use of pharmacologic doses of natural or synthetic ligands. Third: These studies prove the fact that HCA can generate in a short period of time useful information to direct personalized medicine for patients affected by AIS. The extent that these studies can be generalized will be determined by AR HCA studies of additional patients with similar phenotypes.In conclusion these studies have generated several new concepts. More studies are needed with additional cell lines before direct translation into clinical practice. Safety will be an important issue, particularly for the synthetic agonists, which are not FDA-approved. The CAIS phenotype of patients AR-P766S and AR-F764L argues against treating these patients, because 100% of them reared as females maintained throughout life a female gender identity In conclusion, this work represents a step forward toward the utilization of modern high-speed microscopic techniques for the diagnosis and treatment of diseases related to AR. It will be interesting to expand these studies to include evaluation of more global effects of AR rescue using different concentrations of ligand, or with ligands other than the endogenous AR agonists. Future investigation will continue to analyze AR malfunction in AIS and other clinical conditions, such as prostate cancer, hypospadias, cryptorchidism and sarcopenia.


To the Editor: Group A rotaviruses are the most common etiologic agents of acute gastroenteritis in infants and young children, each year resulting in ≈100 million diarrhea episodes and 600,000 deaths worldwide  and the Ethical Committee of the Indonesian National Health Research and Development Institute. Stool specimens and clinical enrollment data were collected for each eligible patient, and all collected items were transported to NAMRU-2 in Jakarta, Indonesia. In December 2007, a stool specimen was collected from a 14-day-old afebrile infant brought to Sumber Waras Hospital in West Jakarta with diarrhea, vomiting, moderate dehydration, and malnutrition. This patient was infected with the rotavirus G12 strain, was hospitalized for 6 days, and was discharged after recovering fully. Bacterial cultures and ova/parasite evaluations were negative for enteric pathogens.,,http://blast.ncbi.nlm.nih.gov) on the National Center for Biological Information website. We then created alignments of nucleotides and deduced amino acid sequences and compared them with a selection of reference strains from the GenBank database. Genetic relationships among G12 were determined by using PAUP version 4.0 beta 10 software (http://paup.csit.fsu.edu). A phylogenetic tree was constructed on the basis of nucleotides 1–971 of the VP7 gene by using the neighbor-joining method and applying the Kimura 2-parameter method with 1,000 bootstrap replicates of the neighbor-joining model.Rotavirus was detected in this specimen and genotyped by multiplex, seminested reverse transcription–PCR (RT-PCR) targeting the VP4 and VP7 genes (The BLASTN search of the VP7 989 nucleotide sequence of the putative G12 Indonesian rotavirus (Indo SWJ0806) showed 98% similarity with published VP7 sequences of rotavirus G12 strains from Japan , Argentina , and Thailand . The Indonesia G12 clustered into the lineage II composed of rotavirus G12 reference strains from Japan, Argentina, South Korea, and Thailand . LineagePhylogenetic analysis showed that the virus clusters into lineage II and that the deduced amino acid sequence is highly conserved compared with other reported rotavirus G12 strains identified. The combination of the P[6] genotype in this rotavirus strain suggests the possibility of a zoonotic transmission (


Mycobacterium avium subsp. paratuberculosis (Map) causes the chronic enteritis called paratuberculosis mainly in cattle, sheep and goats. Evidences that point out an association between Map and Crohn's Disease in humans are increasing. Strain differentiation among Map isolates has proved to be difficult and has limited the study of the molecular epidemiology of paratuberculosis. In order to asses the usefulness of the PCR based short sequence repeat (SSR) analysis of locus 1 and locus 8 in the epidemiological tracing of paratuberculosis strains we here compare for the first time the results of SSR and SnaBI-SpeI pulsed-field gel electrophoresis (PFGE) typing methods in a set of 268 Map isolates from different hosts .SnaBI-SpeI PFGE. SSR type 7_4 was the predominant genotype , but combined with PFGE results the abundance of the most prevalent genotype (7_4&{2-1}) dropped down to 37.7%. SSR types 7_3 and 14_3 were significantly spread amongst isolates recovered from small ruminants. The comparison of SSR1_SSR8 and SnaBI-SpeI PFGE typing of these isolates has shown that both methods perform at similar discriminatory level. These were 0.691 and 0.693, respectively for SSR and PFGE as indicated Simpson's Index of Diversity, and 0.82 when calculated for combined SSR and PFGE genotypes. Overall, SSR1_SSR8 analysis seemed to detect higher levels of within-farm strain diversity and seemed to give higher year-related information. Combination of both typing methods revealed 20 multi-type farms out of the 33 bovine farms studied with more than one isolate.A total of nineteen different multi-locus SSR (SSR1_SSR8) types were identified amongst the 268 isolates compared to the 37 multiplex profiles differentiated by the The particular SSR and PFGE typing approaches described here are in general agreement but they showed some discrepancies that might reflect differing evolutionary processes of Map strains. Both methods are able to reciprocally complement their results and neither should be replaced with the other if sufficient material and time is available. Overall, the results of our comparative analyses suggest that, based on current methodologies available, a combined approach that includes SSR and PFGE seems to provide the highest level of discrimination for Map strain typing with meaningful epidemiological information. Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of paratuberculosis, a chronic digestive disease affecting mainly bovine, ovine, caprine and cervine livestock. Although the aetiology of Crohn's Disease has been subject of strong controversy Simpson's index of diversity = 1-D = 1-[ΣIS and LL carried out the laboratory work, compiled and analysed information and data, and drafted the manuscript. AA, JMG and MVG helped to draft the manuscript. VK and RAJ conceived of the study, and helped to draft the manuscript. All authors read and approved the final manuscript.