Dynamics of CD40 ligand levels in newly diagnosed and treated tuberculosis patients: An evaluation of sCD40L concentration and CD4<sup>+</sup> T cell surface expression

Introduction The HIV/AIDS pandemic has led to a rise in the incidence of tuberculosis and an epidemic of co-infection in many developing countries. Treatment of Mycobacterium tuberculosis in persons with HIV infection presents several challenges to the clinician, particularly in resource-poor countries. As will be discussed in this paper, diagnosis of latent tuberculosis relies on tuberculin skin testing, which has poor sensitivity and reproducibility in immunocompromised patients. The World Health Organization (WHO) recommends treatment of active tuberculosis as the primary means of global tuberculosis control. In practice, treatment of active tuberculosis typically requires that a symptomatic patient self-report to a health service for evaluation and management. Even if this approach to tuberculosis control were sufficient, many logistic and clinical problems remain involving tuberculosis diagnosis and therapy in the patient with HIV/AIDS. Recognizing the significant clinical and public health challenges surrounding the treatment of tuberculosis in patients with HIV infection, this paper will address a number of issues relevant to the care of co-infected patients. These include current guidelines for the treatment of active tuberculosis, as well as the diagnosis and treatment of latent tuberculosis in HIV-positive patients. The paper concludes with a discussion of promising new drugs for tuberculosis treatment. Epidemiology of tuberculosis and HIV co-infection It is estimated that one-third of the world population is infected with M. tuberculosis, the large majority of whom live in the developing world. The HIV pandemic of the past two decades has led to a rise in the incidence of tuberculosis, particularly in sub-Saharan Africa. There is now an emerging pandemic of patients with HIV infection who are co-infected with tuberculosis. As of December 2000, the WHO estimated that approximately 36.1 million persons worldwide are living with HIV and nearly one-third of these persons are co-infected with M. tuberculosis[1]. Approximately 68% of persons co-infected with HIV and tuberculosis live in sub-Saharan Africa, while 22% live in Southeast Asia. In the United States, the Centers for Disease Control and Prevention (CDC) estimates that approximately 40% of new tuberculosis cases among persons aged 15-44 years occur among individuals with HIV infection or AIDS. Tuberculosis rates among HIV-infected individuals in the United States, however, vary significantly among different groups, with highest rates among intravenous drug users and those who are foreign-born. Today, the burden of tuberculosis and HIV infections largely impacts the developing world, as well as the minority and low socio-economic individuals within industrialized countries. This paper will discuss issues in the treatment of M. tuberculosis in patients with HIV/AIDS; however, it is important to recognize that many patients co-infected with HIV and M. tuberculosis have limited or no access to essential diagnostic and therapeutic strategies. Escalating tuberculosis case rates over the past decade are largely attributable to HIV. Immunity to M. tuberculosis is partly under the control of the MHC class II restricted CD4 cells. With the progressive loss of CD4 cells, patients with HIV infection are at increased risk of reactivation of latent tuberculosis, as well as primary tuberculosis infection [2]. In turn, active tuberculosis infection appears to upregulate HIV replication, resulting in further immune compromise and accelerated HIV disease progression [3,4]. As a result, patients with HIV infection and active tuberculosis are at increased risk of opportunistic infections and associated mortality. The case fatality rate by the end of tuberculosis treatment is approximately 20% for new sputum smear-positive cases and up to 50% for new smear-negative cases [5]. Tuberculosis is the leading cause of death among persons with HIV/AIDS worldwide [6]. Guidelines for the treatment of active tuberculosis This section will review recent guidelines for the treatment of tuberculosis published by the Tuberculosis Committee of the Infectious Disease Society of America (IDSA) in conjunction with the American Thoracic Society (ATS) and the CDC [7,8]. In addition, we will review the Directly Observed Treatment Short-course (DOTS) strategy of the WHO for tuberculosis control worldwide. These published guidelines pertain to the treatment of tuberculosis without respect to the patient's HIV status. Modified recommendations for the treatment of tuberculosis in HIV-seropositive patients will be discussed in the following section. In April 2000, the IDSA published practice guidelines for the treatment of tuberculosis [7]. Table 1, adapted from the IDSA publication, lists the 10 essential recommendations for the treatment of patients with tuberculosis. Readers are referred to the original publication for detailed comments pertaining to these recommendations, as well as performance indicators. It should be noted that the IDSA recommendations were developed for use in industrialized nations such as the United States and are currently not feasible in many countries of the world. In Table 1, therefore, we have juxtaposed the WHO DOTS strategy guidelines for management of patients with tuberculosis.Table 1: Infectious Disease Society of America (IDSA) recommendations and World Health Organization Directly Observed Treatment Shortcourse (WHO DOTS) strategy for the management of patients with tuberculosis (TB).In geographic areas where ≥ 4% of the M. tuberculosis isolates are resistant to isoniazid, the IDSA, ATS, and CDC recommend that the usual three-drug regimen of isoniazid, rifampin and pyrazinamide be augmented with a fourth drug, either ethambutol or streptomycin. Clinicians will therefore need to be aware of the susceptibility patterns in their geographic area. In 1997, approximately 84% of the US population lived in states that had ≥ 4% of tuberculosis isolates resistant to isoniazid. Therefore, most patients in the United States should be started on an initial four-drug regimen. This is followed by isoniazid and rifampin for 18 weeks. Although a 6-month course of treatment is recommended, this should be extended to 9 months if there is a delay in AFB, culture conversion or clinical improvement for 8 weeks. The WHO Global Tuberculosis Programme (WHO/GTP) assists over 60 countries with national tuberculosis control and prevention. The priority of the WHO program has been active case finding and cure of infectious tuberculosis cases. In 1993, the WHO/GTP declared tuberculosis a global emergency and began promoting the DOTS strategy. The DOTS strategy consists of five key components: "1) Government commitment to sustained TB control activities; 2) Case detection by sputum smear microscopy among symptomatic patients self-reporting to health services; 3) Standardized treatment regimen of six to eight months for at least all confirmed sputum smear positive cases, with directly observed treatment (DOT) for at least the initial two months; 4) A regular, uninterrupted supply of all essential anti-tuberculosis drugs; and 5) A standardized recording and reporting system that allows assessment of treatment results for each patient and of the TB control programme performance overall" [9] (see Table 1). According to the WHO/GTP, as of 1999, 127 countries had accepted the DOTS strategy and were implementing it to varying degrees [10]. Nevertheless, the WHO/GTP estimate that, in 1999, only 45% of the world population had access to DOTS and 23% of new smear-positive cases were referred to DOTS programs. In addition, there are often a complex array of political, financial, and infrastructure problems that impede local DOTS programs. The current WHO DOTS strategy does not incorporate the diagnosis and treatment of latent tuberculosis. Some experts believe that the DOTS strategy, which focuses exclusively on the treatment of active cases, is insufficient for the control and elimination of tuberculosis, particularly in the HIV/AIDS era [11]. It has been recommended that targeted diagnosis and treatment of latent tuberculosis infection among specific populations be added to national tuberculosis control programs. At present, WHO recommends treatment of latent tuberculosis in HIV-positive patients; but only in settings where it is possible to provide HIV testing and counseling, and where it is possible to exclude cases of active tuberculosis and ensure proper follow-up. The DOTS strategy also employs empiric anti-tuberculous therapy without mycobacterial cultures and drug susceptibility testing. Without drug susceptibility information, it is impossible to identify cases of drug-resistant tuberculosis and to avoid treatment failure and further transmission of drug-resistant strains. The WHO recognizes the threat of multidrug-resistant tuberculosis (MDR-TB) and, in July 1999, convened a working group on DOTS-Plus for the treatment of MDR-TB [12]. DOTS-Plus is a pilot program to provide second-line drugs (i.e., fluoroquinolones, amikacin, kanamycin, capreomycin, cycloserine, para-aminosalicylic acid, and ethionamide) to manage MDR-TB in resource-limited countries. This strategy does not, however, address the role of mycobacterial cultures and drug susceptibility testing for individualized drug therapy and the prevention of drug-resistant tuberculosis. Finally, because DOTS relies on patients self-reporting to health services, additional tuberculosis transmission can take place before the patient is evaluated and receives appropriate therapy. Modeling of the tuberculosis epidemic in Tanzania suggests that DOTS may slow the incidence rate, but in the face of the HIV epidemic is unlikely to reverse the upward trend [13]. Again, some experts advocate targeted case finding, particularly among high-risk groups [11,14]. Diagnosis and treatment of active tuberculosis infection in patients with HIV infection This section deals specifically with the treatment of active tuberculosis in HIV-infected individuals as recommended by the CDC [15,16]. Again, many people co-infected with HIV and tuberculosis in the developing world do not have access to the relevant diagnostic tests and anti-tuberculosis and antiretroviral therapies. To impact tuberculosis-associated morbidity and mortality worldwide, developing countries will require both the skills and commodities to diagnose and treat individuals effectively. At present, tuberculosis treatment for the HIV-positive patient as recommended by the CDC is not applicable in most resource-poor countries. Treatment of active and latent tuberculosis infection in patients with HIV depends on the application of both clinical judgment and appropriate diagnostic tests. Active tuberculosis can occur at any CD4 cell count but atypical presentations are more likely with advanced HIV disease or AIDS. Clinicians should be alert to the sometimes atypical presentations of pulmonary and extrapulmonary tuberculosis in HIV-infected patients. Sputum acid fast staining, mycobacterial cultures, and drug susceptibility testing are recommended in all patients suspected of having tuberculosis. However, patients with HIV are slightly less likely to have positive sputum smears than non-HIV-infected individuals [17]. Likewise, chest radiographic findings can vary depending on the degree of immunosuppression. Patients with CD4 cell counts greater than 200 are more likely to have classic findings of upper lobe infiltrates with cavitary lesions, while those patients with AIDS may more likely have hilar adenopathy and pleural effusions without cavitations [18]. Mycobacteremia and extrapulmonary tuberculosis, especially meningitis and adenopathy, also correlate with diminishing numbers of CD4 cells and degree of immunosuppression. Thus, for patients with HIV infection, the diagnosis of active tuberculosis is more challenging. Clinical suspicion of tuberculosis in a patient known or suspected of being HIV-infected should result in prompt initiation of anti-tuberculosis therapy regardless of sputum staining or radiograph findings. The 1998 CDC recommendations for the treatment of tuberculosis among patients infected with HIV are summarized in Table 2[15]. The treatment of tuberculosis in the HIV-seropositive patient may differ from the standard treatment in the following ways: (i) choice of anti-tubercular regimen and dose adjustments; (ii) duration of treatment (ideally with directly observed therapy); (iii) promotion of antiretroviral therapy; and (iv) monitoring requirements. Due to rapid advances in the management of HIV disease, it is recommended that all patients co-infected with HIV and tuberculosis should be evaluated by a specialist to ensure optimal management.Table 2: Tuberculosis (TB) treatment recommendations for the HIV-seropositive patient.The treatment algorithm begins with establishing the patient's HIV status and whether the patient is on optimal antiretroviral therapy. All patients diagnosed with active tuberculosis should be HIV tested and, if seropositive, evaluated for antiretroviral therapy. It was previously felt that the diagnosis of active tuberculosis should result in the deferral of antiretroviral therapy. Early initiation of antiretroviral therapy is now recommended. While being treated for tuberculosis, the HIV-positive patient not receiving concurrent HIV therapy should be reassessed every 3 months for initiation of antiretroviral therapy. Known or suspected HIV-positive patients should receive prompt initiation of effective anti-tuberculosis therapy. If antiretroviral therapy is not started, the patient can typically receive standard anti-tuberculosis therapy: isoniazid, rifampin, pyrazinamide, and ethambutol. If the patient is to receive simultaneous anti-tuberculosis and antiretroviral therapy, the selected regimens and doses must account for significant drug-drug interactions between the rifamycins (rifampin, rifabutin, rifapentine) and the protease inhibitors and non-nucleoside reverse transcriptase inhibitors (NNRTI). Rifamycins, particularly rifampin, induce the hepatic cytochrome P-450 (CYP450) and reduce the serum levels of protease inhibitors, NNRTI, and other drugs metabolized by the CYP450 system. The CDC generally recommends the substitution of rifabutin, a less potent CYP450 inducer, for rifampin to allow simultaneous use of protease inhibitors and NNRTI. The use of rifampin with protease inhibitors or NNRTI is contraindicated, except in three antiretroviral combinations: (i) the NNRTI efavirenz and two nucleoside reverse transcriptase inhibitors (NRTI); (ii) the protease inhibitor ritonavir and one or more NRTI; and (iii) the combination of ritonavir and saquinavir, either hard-gel or soft-gel capsules [16]. Table 3 lists the recommended anti-tubercular drug doses, depending on frequency of administration and concurrent antiretroviral use.Table 3: Anti-tubercular drug doses.If the HIV-positive patient is already on an effective anti-retroviral regimen at the time of tuberculosis diagnosis, it is desirable to continue the patient on the same antiretroviral regimen with appropriate dose adjustments. If the patient is starting a new antiretroviral regimen, options include: (i) a rifabutin-based regimen with the necessary protease inhibitor or NNRTI dose adjustments; (ii) a non-rifamycin-containing regimen such as isoniazid, streptomycin, pyrazinamide, ethambutol for 2 months, then isoniazid, streptomycin, ethambutol for 7 months; or (iii) a regimen that does not contain a protease inhibitor or NNRTI. Table 4 lists the recommended dose adjustments for rifabutin-based regimens. Given the complexity of these drug interactions, it is recommended that the selection of dual anti-tubercular and antiretroviral therapies be made following consultation with a specialist.Table 4: Protease inhibitor (PI) or non-nucleoside reverse transcriptase inhibitor (NNRTI) dose adjustments with rifabutin.Among the protease inhibitors, ritonavir has the highest potency in inhibiting the CYP450 pathway. With any dose of ritonavir, including low-dose ritonavir 100 mg twice a day, a reduced dose of rifabutin (150 mg two or three times per week) is recommended. According to the 2000 CDC guidelines for the use of rifabutin or rifampin among patients taking protease inhibitors or NNRTI, co-administration of ritonavir with the usual dose of rifampin (600 mg daily or two or three times per week) may be an option but pharmokinetic and clinical data are limited [16]. As previously stated, a patient taking the combination of saquinavir (either the soft-gel or hard-gel capsule) and ritonavir should take the reduced dose of rifabutin (150 mg two or three times per week). The saquinavir-ritonavir combination may possibly be given with the usual dose of rifampin but, again, limited pharmacokinetic and clinical data are available. Saquinavir, as a sole protease inhibitor, is generally not recommended in combination with rifabutin because the serum levels of saquinavir may be decreased as much as 45%. Indinavir, nelfinavir, and amprenavir should not be used in combination with rifampin, but all three protease inhibitors can be administered with a reduced daily dose of rifabutin (150 mg daily) or the usual dose of rifabutin (300 mg two or three times per week). Efavirenz induces CYP450 and accelerates rifamycin metabolism; therefore, efavirenz should be co-administered with an increased dose of rifabutin (450 or 600 mg daily, or 600 mg two or three times per week). According to the recent CDC guidelines, efavirenz may be combined with the usual dose of rifampin (600 mg daily or two or three times per week). The NNRTI nevirapine should typically be given with the usual dose of rifabutin (300 mg daily or two or three times per week). If co-administration of nevirapine with rifampin is clearly indicated, careful monitoring is recommended. The use of delavirdine is contraindicated during the treatment of tuberculosis because the drug levels are markedly decreased with both rifampin and rifabutin. HIV-infected patients have a higher incidence of drug-resistant tuberculosis isolates than non-HIV-infected patients. There have been several reports of increased risk of rifampin resistance among HIV-positive patients [19-21]. Higher rates of drug-resistant tuberculosis in HIV-infected patients may be associated with biological, behavioral, and societal factors including drug malabsorption, non-adherence, nosocomial outbreaks, and inadequate drug therapy in countries with high rates of co-infection. Directly observed therapy, regarded as the best strategy for ensuring adherence and limiting drug-resistant tuberculosis, is recommended for all patients with HIV infection [15]. In a randomized, controlled trial of anti-tuberculosis therapy among HIV-positive patients in Baltimore, Maryland, patients who received supervised therapy for tuberculosis had better survival than those who self-administered therapy [22]. Treatment of drug-resistant tuberculosis involves initiation of a multi-drug regimen tailored to the susceptibility profile of the organism. Due to the diversity of resistance patterns, it is not possible to recommend standardized protocols for therapy. Any regimen should include two or more drugs to which the isolate is susceptible. For HIV-positive patients at risk of MDR-TB, defined as resistance to both isoniazid and rifampin, initial empiric treatment should include second-line tuberculosis drugs to which resistance is uncommon. The 1999 WHO Essential Drug List was to include the following second-line tuberculosis amikacin, kanamycin, capreomycin, cycloserine, acid, and and drug susceptibility testing should be on all tuberculosis isolates and the anti-tuberculosis regimens Although MDR-TB is associated with a high mortality rate especially in resource-poor countries where the detection of drug resistance to months, if at and the second-line drugs are often months is the duration of treatment for pulmonary tuberculosis among HIV-positive patients in the United States a regimen is used [15]. treatment is for patients with a clinical or conversion of sputum cultures from positive to Some experts recommend the use of treatment regimens in all patients with HIV infection, especially among patients with advanced [17]. duration of treatment is clearly recommended in patients with slow clinical or with or HIV-positive individuals with active tuberculosis to be at increased risk of tuberculosis of a 6-month regimen. A trial of isoniazid was among HIV-positive patients in who a 6-month regimen of isoniazid decreased the risk of a of tuberculosis among HIV-positive patients. This not whether these were to M. tuberculosis or both of which have previously been The recommend of isoniazid for HIV-positive patients of tuberculosis therapy. At present, the WHO and the CDC recommend a months of directly observed tuberculosis therapy without Treatment of latent tuberculosis infection in patients with HIV infection It is estimated that 2 people worldwide are infected with latent tuberculosis. Patients with HIV infection are at increased risk of to active disease [2]. In 2000, the and the CDC new guidelines for the diagnosis and treatment of latent tuberculosis infection This public health strategy both prevention by the latent infection before it to active infection and primary prevention by further tuberculosis In addition, the use of active antiretroviral therapy has been to reduce the incidence of tuberculosis among persons with HIV infection The new guidelines recommend targeted of populations and patients at increased risk of tuberculosis infection who from treatment to active for targeted include drug health care of and all patients with HIV Diagnosis of latent tuberculosis infection is on the tuberculin skin new including detection are being developed A has with tuberculin skin testing in are to the diagnostic of this in the United States and in populations with varying degrees of risk for latent tuberculosis. There be two significant of developing tests for the detection of latent M. tuberculosis tests the need for a health service at and their be less than tuberculin skin testing. of latent tuberculosis in an HIV-infected patient can be a diagnostic It has been that the sensitivity of tuberculin skin testing may be in patients with to as well as to and can in persons with HIV infection and of the is associated with higher CD4 cell For these testing is no recommended for the diagnosis of latent tuberculosis. guidelines are to treat latent tuberculosis infection in HIV-infected persons with at high-risk of latent tuberculosis or with recent to a case of active tuberculosis should receive therapy regardless of tuberculin has been the of treatment of latent tuberculosis clinical have a in the risk of to active tuberculosis following months of isoniazid therapy. The optimal duration of isoniazid therapy has also been including the trial of the Tuberculosis and in in the and this or months of isoniazid therapy months of isoniazid therapy reduced the tuberculosis incidence by with for months and 20% for 3 months; however, with months therapy was than for of therapy A recent of isoniazid therapy in that from isoniazid therapy 9 months, with no additional associated with therapy The new and CDC guidelines recommend isoniazid therapy for 9 months (300 mg isoniazid daily mg with months of therapy as a less The to isoniazid therapy include and poor Directly observed therapy has been to adherence but is not important in the treatment of latent tuberculosis has been the of regimens that may adherence and reduce have the of regimens for the treatment of latent tuberculosis in individuals A trial that daily rifampin and pyrazinamide for 2 months was to isoniazid for months for the treatment of latent tuberculosis in HIV-infected persons the of these the new guidelines regimens for the treatment of latent tuberculosis as summarized in Table from their publication rifampin and pyrazinamide for 2 months (600 mg rifampin daily pyrazinamide daily) is the regimen for HIV-infected patients and be for suspected cases of tuberculosis. should be for rifampin in patients receiving protease inhibitors or NNRTI, further is to in the treatment of latent tuberculosis. The same drug-drug interactions and dose adjustments for antiretroviral drugs and rifamycins It is that the also recommends the regimen for individuals the the clinical for this combination have been only in HIV-positive patients. If MDR-TB is the recommended therapy is pyrazinamide and ethambutol or pyrazinamide and a (i.e., or for Treatment for suspected to MDR-TB should be extended to months for HIV-positive American Thoracic Society and Centers for Disease Control and Prevention guidelines for the treatment of latent tuberculosis the use of rifampin pyrazinamide therapy in infected patients has been associated with a rate of patients with resulting from the use of therapy have been to the five of these patients on these CDC and have their 2000 recommendations The (i) that be used with particularly in patients with disease, on or those taking drugs; and (ii) that patients who are treated with be by a health care at and for a of tests and evaluation with a at 8 to treatment therapy with the regimen should be for significant rates of have not been observed in HIV-infected patients treated with this regimen, either in clinical or to the use of the regimen in For HIV-infected patients to persons with active tuberculosis, treatment for latent tuberculosis should be a tuberculin In addition, some experts advocate HIV-infected individuals who in high-risk evaluation for latent tuberculosis treatment should include a careful and and, chest radiograph to exclude active tuberculosis. anti-tubercular drugs in treatment of tuberculosis, particularly for MDR-TB, will on the of new anti-tubercular there are several drugs under that have in M. tuberculosis. In some cases the drug has also in a of tuberculosis, and some have received and Drug for other under include and other and is a rifamycin the serum of which is three times than that of the rifampin The of M. tuberculosis is to or one to that of Given the between and other rifamycin the of over rifampin pharmokinetic In the infected with tuberculosis and in tuberculosis a regimen is less active than a daily regimen, both rifamycins being given at 10 In tuberculosis however, the regimen is significantly less active than the daily regimen In the regimens during the of tuberculosis therapy may provide increased to as well as to health care programs. is an for and other infections that has in M. tuberculosis, to that of and in in a of tuberculosis that at 100 is as as isoniazid at and more than clinical are being to the role of in combination therapy active tuberculosis. and also promising in a of tuberculosis are a class of inhibitors with and The of at 100 is with isoniazid at per an by the and Drug for the treatment of appears to be less active M. tuberculosis than It has been recommended, however, for further at higher is a new to that has been to potent in M. in were also to be to this with of In a administration of this drug at a dose of per led to of disease burden in and with that with isoniazid per and issues in the treatment of tuberculosis in patients with HIV have previously to some of the and issues that the treatment of tuberculosis in resource-poor countries. At the and there may also some important and to effective tuberculosis treatment. A among tuberculosis patients in Tanzania that only of the patients had of the disease and treatment In a recent from people were generally well HIV but tuberculosis of and were to AIDS than tuberculosis. patients with tuberculosis were often as having AIDS. Due to the associated with the that patients with tuberculosis may not or to appropriate These the important impact that the HIV epidemic may have on the public and to tuberculosis. a epidemic of tuberculosis and HIV co-infection in many of the developing world. The increased of tuberculosis and HIV/AIDS and the rise of MDR-TB a health threat to all While significant have been made in developing regimens for the treatment of active and latent tuberculosis, therapy is and second-line drugs are drug resistance is for are the anti-tuberculosis of drugs such as and and the of which may treatment. The optimal use of these drugs in combination therapy is a promising of active In addition, we advocate the of new and tuberculosis drugs that will be made to patients in developing countries.

SIV and HIV Nef proteins disrupt T-cell receptor machinery by down-modulating cell surface expression of CD4 and expression or signaling of CD3-TCR. Nef also down-modulates class I major histocompatibility complex (MHC) surface expression. We show that SIV and HIV-1 Nefs down-modulate CD28, a major co-stimulatory receptor that mediates effective T-cell activation, by accelerating CD28 endocytosis. The effects of Nef on CD28, CD4, CD3 and class I MHC expression are all genetically separable, indicating that all are selected independently. In cells expressing a Nef-green fluorescent protein (GFP) fusion, CD28 co-localizes with the AP-2 clathrin adaptor and Nef-GFP. Mutations that disrupt Nef interaction with AP-2 disrupt CD28 down-regulation. Furthermore, HIV and SIV Nefs use overlapping but distinct target sites in the membrane-proximal region of the CD28 cytoplasmic domain. Thus, Nef probably induces CD28 endocytosis via the AP-2 pathway, and this involves a ternary complex containing Nef, AP-2 and CD28. The likely consequence of the concerted down-regulation of CD28, CD4 and/or CD3 by Nef is disruption of antigen-specific signaling machineries in infected T cells following a productive antigen recognition event.

Functional Role of the C Terminus of Human Organic Anion Transporter hOAT1

Background: Early diagnosis and prompt initiation of treatment is an essential component of revised National Tuberculosis Control Programme. Any type of delay (patient delay, health system delay) may worsen the disease, increase the risk of death and enhance disease transmission in the community. The study intended to know the extent of patient delay and health system delay in diagnosis and treatment of tuberculosis (TB) patients and to assess factors associated with patient and health system delay.Methods:A cross sectional study on 217 new smear positive tuberculosis patients aged above 15 years who were registered between Nov-2008 to Sept-2009 for DOTS under RNTCP in Mysore tuberculosis unit was included in the study. Interview was conducted using pre tested semi structured questionnaire within 2 months of diagnosis to minimize recall bias.Results: The median delay among 217 participants was 27 days due to patient delay, 17 days due to health system and 49 days in total delay respectively. The delay in seeking care was more than one month in 39% patients. In 44% of patients there was more than 15 days delay in the diagnosis by the health care providers. In 10% of patients treatment was not initiated within 7 days of diagnosis.Conclusions:More specific and effective health education to the general public on seeking of appropriate medial consultation through proper channel is likely to decrease patient delay. Continuing medical education for private practitioners is needed to encourage vigilance for TB and earlier use of diagnostic tests.

The Ministry of Health in China has enacted a revised National Tuberculosis Control Program (NTP) guidelines and clinical pathway (CP) on new smear-positive pulmonary tuberculosis (TB) inpatients to improve the quality of TB care and asked doctors' compliance to them on the treatment of TB inpatients. However, it remains unknown whether doctors adhere to them well. So this study focuses on evaluating the doctors' compliance with them in one representative TB hospital for medical quality improvement. A hospital-based retrospective study involving all medical records of newly diagnosed smear-positive pulmonary TB inpatients from July 2011 to July 2013. Analysis indicators including adequate drug regimens rate, adequate drug dosages rate and adequate length of hospital stay rate were chosen to assess doctors' compliance with NTP guidelines and CP on the treatment of new smear-positive pulmonary TB inpatients. The optimal value of these indicators is 100%. Of the 334 inpatients selected, the rate of adequate drug regimens prescribed is 26.95% (90/334), and the rate of adequate drug dosages is 0% (0/90). For the dosage of single drug, the rates of adequate dosage of isoniazid, rifampicin, pyrazinamide and ethambutol are 24.44% (22/90), 85.56% (77/90), 70% (63/90) and 13.33% (12/90). Moreover, 75.56% (68/90) of isoniazid was prescribed too high and 83.34% (75/90) of ethambutol was prescribed too low. The rate of adequate length of hospital stay provided is 28.44% (95/334). Doctors' compliance with NTP guidelines and CP on new smear-positive pulmonary TB inpatients is depressed and needs improvement.

Plasma retinol-binding protein (RBP), the principal carrier of vitamin A in the blood, delivers vitamin A from liver, the site of storage, to distant organs that need vitamin A, such as the eye, brain, placenta, and testis. STRA6 is a high-affinity membrane receptor for RBP and mediates vitamin A uptake in these target organs. STRA6 is a 74-kDa multi-transmembrane domain protein that represents a new class of membrane transport protein. In this study, we used an unbiased strategy by analyzing >900 random mutants of STRA6 to study its structure and function, and we identified an essential RBP-binding domain in STRA6. Mutations in any of the three essential residues in this domain can almost completely abolish binding of STRA6 to RBP and its vitamin A uptake activity from holo-RBP without affecting its cell surface expression. We have also functionally characterized the mutations in human STRA6 that cause severe birth defects as well as several human polymorphisms. All STRA6 mutants associated with severe birth defects have largely abolished vitamin A uptake activity, consistent with the severe clinical phenotypes. In addition, we have identified a human polymorphism that significantly reduces the vitamin A uptake activity of STRA6. Interestingly, the residue affected by this polymorphism is located in the RBP-binding domain we identified, and the polymorphism causes decreased vitamin A uptake by reducing RBP binding. This study identifies an essential functional domain in STRA6 and a human polymorphism in this domain that leads to reduced vitamin A uptake activity.

Host-directed-therapy strategies are warranted to fight tuberculosis. Here we assess the safety and immunogenicity of adjunctive vaccination with the H56:IC31 candidate and cyclooxygenase-2-inhibitor treatment (etoricoxib) in pulmonary and extra-pulmonary tuberculosis patients in a randomized open-label phase I/II clinical trial (TBCOX2, NCT02503839). A total of 222 patients were screened, 51 enrolled and randomized; 13 in the etoricoxib-group, 14 in the H56:IC31-group, 12 in the etoricoxib+H56:IC31-group and 12 controls. Three Serious Adverse Events were reported in the etoricoxib-groups; two urticarial rash and one possible disease progression, no Serious Adverse Events were vaccine related. H56:IC31 induces robust expansion of antigen-specific T-cells analyzed by fluorospot and flow cytometry, and higher proportion of seroconversions. Etoricoxib reduced H56:IC31-induced T-cell responses. Here, we show the first clinical data that H56:IC31 vaccination is safe and immunogenic in tuberculosis patients, supporting further studies of H56:IC31 as a host-directed-therapy strategy. Although etoricoxib appears safe, our data do not support therapy with adjunctive cyclooxygenase-2-inhibitors.

ObjectiveThe objective of this study is to assess effectiveness of directly observed treatment short course (DOTS) in treatment of tuberculosis (TB) patients in all public health facilities of Debre Tabor town, Ethiopia from January 2016 to December 2017.ResultAmong 354 TB patients, 53.1% were males. Furthermore 22.6%, 40.4%, and 37% were smear positive pulmonary, smear negative pulmonary and extra pulmonary TB respectively. Study also revealed that TB–human immunodeficiency virus (HIV) co-infection and overall TB treatment success rate were 18.1% and 90.7% respectively. Regular weigh follow-up, sputum follow-up and HIV status were significantly associated with treatment success with P-value < 0.001, < 0.001 and 0.334 respectively. But TB treatment success weren’t associated with sex (P = 8.62), health facility type (P = 0.749) and TB type (P = 0.778). The study also showed that the overall TB treatment success rate was in line with World Health Organization (WHO) target on treatment success rate. Furthermore the study indicated higher TB–HIV co-infection and variations in conducting regular weight and sputum follow-up among HFs.

BackgroundTreatment delay is an important indicator of access to tuberculosis diagnosis and treatment. Analyses of patient delay (i.e. time interval between onset of symptoms and first consultation of a health care provider) and health care delay (i.e. time interval between first consultation and start of treatment) can inform policies to improve access. This study assesses the patient, health care provider and total delay in diagnosis and treatment of new smear-positive pulmonary tuberculosis patients, and the risk factors for long delay, in Vietnam.MethodsA cross-sectional survey of new patients treated by the National Tuberculosis Control Programme was conducted in 70 randomly selected districts in Vietnam. All consecutively registered patients in one quarter of 2002 were interviewed using a pre-coded structured questionnaire.ResultsMedian (range) delay was 4 weeks (1–48) for total, 3 (1–48) weeks for patient and 1 (0–25) week for health care delay. Patients with long total delay (≥ 12 weeks, 15%) accounted for 49% of the cumulative number of delay-weeks. Independent risk factors (p < 0.05) for long total delay were female sex, middle age, remote setting, residence in the northern or central area, and initial visit to the private sector. For long patient delay (≥ 6 weeks) this was female sex, belonging to an ethnic minority, and living at > 5 km distance from a health facility or in the northern area. For long health care delay (≥ 6 weeks) this was urban setting, residence in the central area and initial visit to a communal health post, TB hospital or the private sector.ConclusionAnalyses of patient and treatment delays can indicate target groups and areas for health education and strengthening of the referral system, in particular between the private sector and the NTP.

The Ca(2+)-sensing receptor (CaR) regulates salt and water transport in the kidney as demonstrated by the association of gain of function CaR mutations with a Bartter syndrome-like, salt-wasting phenotype, but the precise mechanism for this effect is not fully established. We found previously that the CaR interacts with and inactivates an inwardly rectifying K(+) channel, Kir4.1, which is expressed in the distal nephron that contributes to the basolateral K(+) conductance, and in which loss of function mutations are associated with a complex phenotype that includes renal salt wasting. We now find that CaR inactivates Kir4.1 by reducing its cell surface expression. Mutant CaRs reduced Kir4.1 cell surface expression and current density in HEK-293 cells in proportion to their signaling activity. Mutant, activated Gα(q) reduced cell surface expression and current density of Kir4.1, and these effects were blocked by RGS4, a protein that blocks signaling via Gα(i) and Gα(q). Other α subunits had insignificant effects. Knockdown of caveolin-1 blocked the effect of Gα(q) on Kir4.1, whereas knockdown of the clathrin heavy chain had no effect. CaR had no comparable effect on the renal outer medullary K(+) channel, an apical membrane distal nephron K(+) channel that is internalized by clathrin-coated vesicles. Co-immunoprecipitation studies showed that the CaR and Kir4.1 physically associate with caveolin-1 in HEK cells and in kidney extracts. Thus, the CaR decreases cell surface expression of Kir4.1 channels via a mechanism that involves Gα(q) and caveolin. These results provide a novel molecular basis for the inhibition of renal NaCl transport by the CaR.

Nicotinic acetylcholine receptor (nAChR) cell surface expression levels are modulated during nicotine dependence and multiple disorders of the nervous system, but the mechanisms underlying nAChR trafficking remain unclear. To determine the role of cysteine residues, including their palmitoylation, on neuronal α4 nAChR subunit maturation and cell surface trafficking, the cysteines in the two intracellular regions of the receptor were replaced with serines using site-directed mutagenesis. Palmitoylation is a post-translational modification that regulates membrane receptor trafficking and function. Metabolic labeling with [(3)H]palmitate determined that the cysteine in the cytoplasmic loop between transmembrane domains 1 and 2 (M1-M2) is palmitoylated. When this cysteine is mutated to a serine, producing a depalmitoylated α4 nAChR, total protein expression decreases, but surface expression increases compared with wild-type α4 levels, as determined by Western blotting and enzyme-linked immunoassays, respectively. The cysteines in the M3-M4 cytoplasmic loop do not appear to be palmitoylated, but replacing all of the cysteines in the loop with serines increases total and cell surface expression. When all of the intracellular cysteines in both loops are mutated to serines, there is no change in total expression, but there is an increase in surface expression. Calcium accumulation assays and high affinity binding for [(3)H]epibatidine determined that all mutants retain functional activity. Thus, our results identify a novel palmitoylation site on cysteine 273 in the M1-M2 loop of the α4 nAChR and determine that cysteines in both intracellular loops are regulatory factors in total and cell surface protein expression of the α4β2 nAChR.

Neural adhesion molecule NB-3 plays an important role in the apical dendrite development of layer V pyramidal neurons in the visual cortex, and receptor-like protein-tyrosine phosphatase α (PTPα) mediates NB-3 signaling in this process. Here we investigated the role of PTPα in regulating cell surface expression of NB-3. We found that cortical neurons from PTPα knock-out mice exhibited a lower level of NB-3 at the cell surface. When expressed in COS1 cells, NB-3 was enriched in the Golgi apparatus with a low level of cell surface expression. However, co-expression of PTPα increased the cell surface distribution of NB-3. Further analysis showed that PTPα facilitated Golgi exit of NB-3 and stabilized NB-3 protein at the cell surface by preventing its release from the plasma membrane. The extracellular region of PTPα but not its catalytic activity is necessary for its effect on NB-3 expression. Thus, the PTPα-mediated increase of NB-3 level at the cell surface represents a novel function of PTPα in NB-3 signaling in neural development.

Mutations in SCN1A, encoding the voltage-gated sodium channel Na(V)1.1, are the most common cause of severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome. SMEI is most often associated with premature truncations of Na(V)1.1 that cause loss of function, but nontruncating mutations also occur. We hypothesized that some nontruncating mutations might impair trafficking of Na(V)1.1 to the plasma membrane. Here we demonstrated that seven nontruncating missense or in-frame deletion mutations (L986F, delF1289, R1648C, F1661S, G1674R, and G1979E) exhibited reduced cell surface expression relative to wild type (WT) Na(V)1.1 consistent with impaired trafficking. We tested whether two commonly prescribed antiepileptic drugs (phenytoin, lamotrigine), as well as the cystic fibrosis transmembrane conductance regulator (CFTR) trafficking corrector VRT-325, could rescue cell surface and functional expression of two representative Na(V)1.1 mutants (R1648C, G1674R). Treatment of cells with phenytoin increased cell surface expression of WT-Na(V)1.1 and both mutant channels, whereas lamotrigine only increased surface expression of R1648C. VRT-325 did not alter surface expression of WT-Na(V)1.1 or mutant channels. Although phenytoin increased surface expression of G1674R, channel function was not restored, suggesting that this mutation also causes an intrinsic loss of function. Both phenytoin and lamotrigine increased functional expression of R1648C, but lamotrigine also increased persistent sodium current evoked by this mutation. Our findings indicate that certain nontruncating SCN1A mutations associated with SMEI have impaired cell surface expression and that some alleles may be amenable to pharmacological rescue of this defect. However, rescue of dysfunctional Na(V)1.1 channels to the plasma membrane could contribute to exacerbating rather than ameliorating the disease.

Recent work putatively linked a rare genetic variant of the chaperone Resistant to Inhibitors of acetylcholinesterase (RIC3) (NM_024557.4:c.262G > A, NP_078833.3:p.G88R) to a unique ability to speak backwards, a language skill that is associated with exceptional working memory capacity. RIC3 is important for the folding, maturation, and functional expression of α7 nicotinic acetylcholine receptors (nAChR). We compared and contrasted the effects of RIC3G88R on assembly, cell surface expression, and function of human α7 receptors using fluorescent protein tagged α7 nAChR and Förster resonance energy transfer (FRET) microscopy imaging in combination with functional assays and 125I-α-bungarotoxin binding. As expected, the wild-type RIC3 protein was found to increase both cell surface and functional expression of α7 receptors. In contrast, the variant form of RIC3 decreased both. FRET analysis showed that RICG88R increased the interactions between RIC3 and α7 protein in the endoplasmic reticulum. These results provide interesting and novel data to show that a RIC3 variant alters the interaction of RIC3 and α7, which translates to decreased cell surface and functional expression of α7 nAChR.

Development of CART model for prediction of tuberculosis treatment loss to follow up in the state of São Paulo, Brazil: A case–control study