Correction: Comparison of artemether-lumefantrine and chloroquine with and without primaquine for the treatment of Plasmodium vivax infection in Ethiopia: A randomized controlled trial.

[This corrects the article DOI: 10.1371/journal.pmed.1002299.].

The effect of holes in long-lasting insecticidal nets on malaria in Malawi: results from a case-control study.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are a cornerstone of malaria prevention. Holes develop in LLINs over time and compromise their physical integrity, but how holes affect malaria transmission risk is not well known. METHODS: After a nationwide mass LLIN distribution in July 2012, a study was conducted to assess the relationship between LLIN damage and malaria. From March to September 2013, febrile children ages 6-59 months who consistently slept under LLINs (every night for 2 weeks before illness onset) were enrolled in a case-control study at Machinga District Hospital outpatient department. Cases were positive for Plasmodium falciparum asexual parasites by microscopy while controls were negative. Digital photographs of participants' LLINs were analysed using an image-processing programme to measure holes. Total hole area was classified by quartiles and according to the World Health Organization's proportionate hole index (pHI) cut-offs [< 79 cm2 (good), 80-789 cm2 (damaged), and > 790 cm2 (too torn)]. Number of holes by location and size, and total hole area, were compared between case and control LLINs using non-parametric analyses and logistic regression. RESULTS: Of 248 LLINs analysed, 97 (39%) were from cases. Overall, 86% of LLINs had at least one hole. The median number of holes of any size was 9 [interquartile range (IQR) 3, 22], and most holes were located in the lower halves of the nets [median 7 (IQR 2, 16)]. There were no differences in number or location of holes between LLINs used by cases and controls. The median total hole area was 10 cm2 (IQR 2, 125) for control LLINs and 8 cm2 (IQR 2, 47) for case LLINs (p = 0.10). Based on pHI, 109 (72%) control LLINs and 83 (86%) case LLINs were in "good" condition. Multivariable modeling showed no association between total hole area and malaria, controlling for child age, caregiver education, and iron versus thatched roof houses. CONCLUSIONS: LLIN holes were not associated with increased odds of malaria in this study. However, most of the LLINs were in relatively good condition 1 year after distribution. Future studies should examine associations between LLIN holes and malaria risk with more damaged nets.

Comparison of artemether-lumefantrine and chloroquine with and without primaquine for the treatment of Plasmodium vivax infection in Ethiopia: A randomized controlled trial.

BACKGROUND: Recent efforts in malaria control have resulted in great gains in reducing the burden of Plasmodium falciparum, but P. vivax has been more refractory. Its ability to form dormant liver stages confounds control and elimination efforts. To compare the efficacy and safety of primaquine regimens for radical cure, we undertook a randomized controlled trial in Ethiopia. METHODS AND FINDINGS: Patients with normal glucose-6-phosphate dehydrogenase status with symptomatic P. vivax mono-infection were enrolled and randomly assigned to receive either chloroquine (CQ) or artemether-lumefantrine (AL), alone or in combination with 14 d of semi-supervised primaquine (PQ) (3.5 mg/kg total). A total of 398 patients (n = 104 in the CQ arm, n = 100 in the AL arm, n = 102 in the CQ+PQ arm, and n = 92 in the AL+PQ arm) were followed for 1 y, and recurrent episodes were treated with the same treatment allocated at enrolment. The primary endpoints were the risk of P. vivax recurrence at day 28 and at day 42. The risk of recurrent P. vivax infection at day 28 was 4.0% (95% CI 1.5%-10.4%) after CQ treatment and 0% (95% CI 0%-4.0%) after CQ+PQ. The corresponding risks were 12.0% (95% CI 6.8%-20.6%) following AL alone and 2.3% (95% CI 0.6%-9.0%) following AL+PQ. On day 42, the risk was 18.7% (95% CI 12.2%-28.0%) after CQ, 1.2% (95% CI 0.2%-8.0%) after CQ+PQ, 29.9% (95% CI 21.6%-40.5%) after AL, and 5.9% (95% CI 2.4%-13.5%) after AL+PQ (overall p < 0.001). In those not prescribed PQ, the risk of recurrence by day 42 appeared greater following AL treatment than CQ treatment (HR = 1.8 [95% CI 1.0-3.2]; p = 0.059). At the end of follow-up, the incidence rate of P. vivax was 2.2 episodes/person-year for patients treated with CQ compared to 0.4 for patients treated with CQ+PQ (rate ratio: 5.1 [95% CI 2.9-9.1]; p < 0.001) and 2.3 episodes/person-year for AL compared to 0.5 for AL+PQ (rate ratio: 6.4 [95% CI 3.6-11.3]; p < 0.001). There was no difference in the occurrence of adverse events between treatment arms. The main limitations of the study were the early termination of the trial and the omission of haemoglobin measurement after day 42, resulting in an inability to estimate the cumulative risk of anaemia. CONCLUSIONS: Despite evidence of CQ-resistant P. vivax, the risk of recurrence in this study was greater following treatment with AL unless it was combined with a supervised course of PQ. PQ combined with either CQ or AL was well tolerated and reduced recurrence of vivax malaria by 5-fold at 1 y. TRIAL REGISTRATION: ClinicalTrials.gov NCT01680406.

The effectiveness of long-lasting, insecticide-treated nets in a setting of pyrethroid resistance: a case-control study among febrile children 6 to 59 months of age in Machinga District, Malawi.

BACKGROUND: The escalating level of mosquito resistance to pyrethroid insecticides threatens the effectiveness of insecticide-treated nets (ITNs) for malaria control in Malawi. An evaluation of the effectiveness of ITNs for preventing malaria in children aged 6-59 months old, after 1 year of mass distribution of LLINs was conducted in Machinga District, Malawi, an area of moderate pyrethroid resistance. METHODS: A facility-based, case-control study among children 6-59 months was conducted in an area of pyrethroid resistance between March and September 2013 in Machinga District. Cases and controls were children with fever who sought care from the same hospital and tested positive and negative, respectively, for malaria parasites by microscopy. RESULTS: A high proportion of both cases (354 of 404 or 87.6 %) and controls (660 of 778 or 84.8 %) slept under an ITN the night before the survey. In univariable logistic regression, older age (24-59 months versus 6-23 months, p < 0.001), sleeping on the floor versus a mattress (p < 0.001), and open versus closed house eaves (p = 0.001) were associated with increased odds of malaria, whilst secondary education of the caretaker, having windows on multiple walls, and being in the least poor wealth quintile (p < 0.001 for each) reduced the odds of malaria; ITN use was not associated with malaria (p = 0.181). In multivariable analysis, older age (p < 0.001) and secondary education of the caregiver (p = 0.011) were the only factors significantly associated with malaria. CONCLUSION: This study did not find a significant personal protective effect of ITNs. However, high use of ITNs in the community and recent findings of lower malaria incidence in ITN users compared to bed net non-users from a cohort study in the same area suggest that ITNs provide community protection to both users and non-users alike in this area.

Evaluation of case management of uncomplicated malaria in Haiti: a national health facility survey, 2012.

BACKGROUND: Malaria is a public health concern in Haiti, although there are limited data on its burden and case management. National malaria guidelines updated in 2012 recommend treatment with chloroquine and primaquine. In December 2012, a nationally-representative cross-sectional survey of health facilities (HFs) was conducted to determine malaria prevalence among febrile outpatients and malaria case management quality at baseline before scale-up of diagnostics and case management training. METHODS: Among all 833 HFs nationwide, 30 were selected randomly, in proportion to total HFs per region, for 2-day evaluations. Survey teams inventoried HF material and human resources. Outpatients of all ages were screened for temperature >37.5 °C or history of fever; those without severe symptoms were consented and enrolled. Providers evaluated and treated enrolled patients according to HF standards; the survey teams documented provider-ordered diagnostic tests and treatment decisions. Facility-based test results [microscopy and malaria rapid diagnostic tests (RDTs)] were collected from HF laboratories. Blood smears for gold-standard microscopy, and dried blood spots for polymerase chain reaction (PCR) were obtained. RESULTS: Malaria diagnostic capacity, defined as completing a test for an enrolled patient or having adequate resources for RDTs or microscopy, was present in 11 (37 %) HFs. Among 459 outpatients screened, 257 (56 %) were febrile, of which 193 (75 %) were eligible, and 153 (80 %) were enrolled. Among 39 patients with facility-level malaria test results available on the survey day, 11 (28 %) were positive, of whom 6 (55 %) were treated with an anti-malarial. Twenty-seven (95 %) of the 28 patients testing negative were not treated with an anti-malarial. Of 114 patients without test results available, 35 (31 %) were presumptively treated for malaria. Altogether, 42 patients were treated with an anti-malarial, one (2 %) according to Haiti's 2012 guidelines. Of 140 gold-standard smears, none were positive, although one patient tested positive by PCR, a more sensitive technique. The national prevalence of malaria among febrile outpatients is estimated to be 0.5 % (95 % confidence interval 0-1.7 %). CONCLUSIONS: Malaria is an uncommon cause of fever in Haitian outpatients, and limited, often inaccurate, diagnostic capacity at baseline contributes to over diagnosis. Scale-up of diagnostics and training on new guidelines should improve malaria diagnosis and treatment in Haiti.

Adherence to malaria prophylaxis among Peace Corps Volunteers in the Africa region, 2013.

BACKGROUND: Although malaria can be prevented with prophylaxis, it is diagnosed in over 100 Africa-region Peace Corps Volunteers annually. This suggests that prophylaxis non-adherence is a problem in these non-immune travelers. METHODS: We investigated Volunteers' knowledge, attitudes, and practices regarding prophylaxis using an internet-based survey during August 19-September 30, 2013. Adherence was defined as taking doxycycline or atovaquone-proguanil daily, or taking mefloquine doses no more than 8 days apart. RESULTS: The survey was sent to 3248 Volunteers. Of 781 whose responses were analyzed, 514 (73%) reported adherence to prophylaxis. The most common reasons for non-adherence were forgetting (n = 530, 90%); fear of long-term adverse effects (LTAEs; n = 316, 54%); and experiencing adverse events that Volunteers attributed to prophylaxis (n = 297, 51%). Two hundred fourteen (27%) Volunteers reported not worrying about malaria. On multivariate analysis controlling for sex and experiencing adverse events Volunteers attributed to prophylaxis, the factor most strongly associated with non-adherence was being prescribed mefloquine (OR 5.4, 95% confidence interval 3.2-9.0). CONCLUSIONS: We found moderate adherence and a prevailing fear of LTAEs among Volunteers. Strategies to improve prophylaxis adherence may include medication reminders, increasing education about prophylaxis safety and malaria risk, and promoting prompt management of prophylaxis side effects.

Knowledge, attitudes, and practices regarding antimalarial chemoprophylaxis in U.S. Peace Corps Volunteers--Africa, 2013.

Long-term travelers to areas where malaria is endemic are at risk for this potentially fatal disease; however, malaria can be prevented through the use of insecticide-treated bednets, mosquito repellents, and chemoprophylaxis. Three options for chemoprophylaxis are available in the Africa region: mefloquine, doxycycline, and atovaquone-proguanil. These options differ by dosing regimen, cost, and side effect profile. Long-term adverse effects of these drugs have been reported rarely.

Changes in HIV risk behavior and seroincidence among clients presenting for repeat HIV counseling and testing in Moshi, Tanzania.

While HIV counseling and testing (HCT) has been considered an HIV preventive measure in Africa, data are limited describing behavior changes following HCT. This study evaluated behavior changes and estimated HIV seroincidence rate among returning HCT clients. Repeat and one-time testing clients receiving HCT services in Moshi, Tanzania were identified. Information about sociodemographic characteristics, HIV-related behaviors and testing reasons were collected, along with HIV serostatus. Six thousand seven hundred and twenty-seven clients presented at least once for HCT; 1235 (18.4%) were HIV seropositive, median age was 29.7 years and 3712 (55.3%) were women. 1382 repeat and 4272 one-time testers were identified. Repeat testers were more likely to be male, older, married, or widowed, and testing because of unfaithful partner or new sexual partner. One-time testers were more likely to be students and testing due to illness. At second test, repeat testers were more likely to report that partners had received HIV testing, not have concurrent partners, not suspect partners have HIV, and have partners who did not have other partners. Clients who intended to change behaviors after the first test were more likely to report having changed behaviors by remaining abstinent (OR 2.58; p<0.0001) or using condoms (OR 2.00; p=0.006) at the second test. HIV seroincidence rate was 1.49 cases/100 person-years (PY). Clients presenting for repeat HCT reported some reduction of risky behavior and improved knowledge of sexual practices and HIV serostatus of their partners. Promoting behavior change through HCT should continue to be a focus of HIV prevention efforts in sub-Saharan Africa.

Total lymphocyte count and World Health Organization pediatric clinical stage as markers to assess need to initiate antiretroviral therapy among human immunodeficiency virus-infected children in Moshi, Northern Tanzania.

BACKGROUND: The World Health Organization (WHO) has recommended the use of clinical staging alone and with total lymphocyte count to identify HIV infected children in need of antiretroviral therapy (ART) in resource-limited settings, when CD4 cell count is not available. METHODS: We prospectively enrolled children obtaining care for HIV infection at the Kilimanjaro Christian Medical Centre Pediatric Infectious Diseases Clinic in Moshi, Tanzania between March 2004 and May 2006 for this cohort study. RESULTS: One hundred ninety two (89.7%) of 214 children met WHO ART initiation criteria based on clinical staging or CD4 cell count. Several low-cost measures identified individuals who met WHO ART initiation criteria to the following degree: WHO stages 3 or 4 had 87.5% (95% CI, 82.8-92.1) sensitivity and, by definition, 100% (CI, 100-100) specificity; WHO recommended advance disease TLC cutoffs: sensitivity = 23.9% (95% CI, 17.3-30.5) specificity = 78.2% (95% CI, 67.3-89.1). Low TLC was a common finding, (50 of 214; 23%); however, it did not improve the sensitivity or specificity of clinical staging in identifying the severely immunosuppressed stage 2 children. Growth failure or use of total lymphocyte counts in isolation were not reliable indicators of severe immunosuppression or need to initiate ART. CONCLUSION: The use of total lymphocyte count does not improve the ability to identify children in need of ART compared with clinical staging alone. Low absolute lymphocyte count did not correlate with severe immunosuppression based on CD4 cell count in this cohort.

Gender differences in the risk of HIV infection among persons reporting abstinence, monogamy, and multiple sexual partners in northern Tanzania.

BACKGROUND: Monogamy, together with abstinence, partner reduction, and condom use, is widely advocated as a key behavioral strategy to prevent HIV infection in sub-Saharan Africa. We examined the association between the number of sexual partners and the risk of HIV seropositivity among men and women presenting for HIV voluntary counseling and testing (VCT) in northern Tanzania. METHODOLOGY/ PRINCIPAL FINDINGS: Clients presenting for HIV VCT at a community-based AIDS service organization in Moshi, Tanzania were surveyed between November 2003 and December 2007. Data on sociodemographic characteristics, reasons for testing, sexual behaviors, and symptoms were collected. Men and women were categorized by number of lifetime sexual partners, and rates of seropositivity were reported by category. Factors associated with HIV seropositivity among monogamous males and females were identified by a multivariate logistic regression model. Of 6,549 clients, 3,607 (55%) were female, and the median age was 30 years (IQR 24-40). 939 (25%) females and 293 (10%) males (p<0.0001) were HIV seropositive. Among 1,244 (34%) monogamous females and 423 (14%) monogamous males, the risk of HIV infection was 19% and 4%, respectively (p<0.0001). The risk increased monotonically with additional partners up to 45% (p<0.001) and 15% (p<0.001) for women and men, respectively with 5 or more partners. In multivariate analysis, HIV seropositivity among monogamous women was most strongly associated with age (p<0.0001), lower education (p<0.004), and reporting a partner with other partners (p = 0.015). Only age was a significant risk factor for monogamous men (p = 0.0004). INTERPRETATION: Among women presenting for VCT, the number of partners is strongly associated with rates of seropositivity; however, even women reporting lifetime monogamy have a high risk for HIV infection. Partner reduction should be coupled with efforts to place tools in the hands of sexually active women to reduce their risk of contracting HIV.

Predictors of incomplete adherence, virologic failure, and antiviral drug resistance among HIV-infected adults receiving antiretroviral therapy in Tanzania.

BACKGROUND: Access to antiretroviral therapy is rapidly expanding in sub-Saharan Africa. Identifying the predictors of incomplete adherence, virologic failure, and antiviral drug resistance is essential to achieving long-term success. METHODS: A total of 150 subjects who had received antiretroviral therapy for at least 6 months completed a structured questionnaire and adherence assessment, and plasma human immunodeficiency virus (HIV) RNA levels were measured. Virologic failure was defined as an HIV RNA level >400 copies/mL; for patients with an HIV RNA level >1000 copies/mL, genotypic antiviral drug resistance testing was performed. Predictors were analyzed using bivariable and multivariable logistic regression models. RESULTS: A total of 23 (16%) of 150 subjects reported incomplete adherence. Sacrificing health care for other necessities (adjusted odds ratio [AOR], 19.8; P<.01) and the proportion of months receiving self-funded treatment (AOR, 23.5; P=.04) were associated with incomplete adherence. Virologic failure was identified in 48 (32%) of 150 subjects and was associated with incomplete adherence (AOR, 3.6; P=.03) and the proportion of months receiving self-funded antiretroviral therapy (AOR, 13.0; P=.02). Disclosure of HIV infection status to family members or others was protective against virologic failure (AOR, 0.10; P=.04). CONCLUSIONS: Self-funded treatment was associated with incomplete adherence and virologic failure, and disclosure of HIV infection status was protective against virologic failure. Efforts to provide free antiretroviral therapy and to promote social coping may enhance adherence and reduce rates of virologic failure.

Predicting CD4 lymphocyte count <200 cells/mm(3) in an HIV type 1-infected African population.

Clinical criteria are recommended to select HIV-infected patients for initiation of antiretroviral therapy when CD4 lymphocyte testing is unavailable. We evaluated the performance characteristics of WHO staging criteria, anthropometrics, and simple laboratory measurements for predicting CD4 lymphocyte count (CD4 count) <200 cells/mm(3) among HIV-infected patients in Tanzania. A total of 202 adults, diagnosed with HIV infection through community-based testing, underwent a detailed evaluation including staging history and examination, anthropometry, complete blood count, erythrocyte sedimentation rate (ESR), and CD4 count. Univariable analysis and recursive partitioning were used to identify characteristics associated with CD4 count 200 cells/mm(3). Of 202 participants 109 (54%) had a CD4 count <200 cells/mm(3). Characteristics most strongly associated with CD4 count <200 cells/mm(3) (p-value <0.0001) were the presence of mucocutaneous manifestations (72% vs. 28%), lower total lymphocyte count (TLC) (median 1,450 vs. 2,200 cells/mm(3)), lower total white blood cell count (median 4,200 vs. 5,500 cells/mm(3)), and higher ESR (median 95 vs. 53 mm/h). In a partition tree model, TLC <1,200 cells/mm(3), ESR >or=120 mm/h, or the presence of mucocutaneous manifestations yielded a sensitivity of 0.85 and specificity of 0.63 for predicting CD4 count <200 cells/mm(3). The sensitivity of the 2006 WHO Staging system improved from 0.75 to 0.93 with inclusion of these parameters, at the expense of specificity (0.36 to 0.26). The presence of mucocutaneous manifestations, TLC <1,200 cells/mm(3), or ESR >or=120 mm/h was a strong predictor of CD4 count <200 cells/mm(3) and enhanced the sensitivity of the 2006 WHO staging criteria for identifying patients likely to benefit from antiretrovirals.

Antiretroviral treatment literacy among HIV voluntary counseling and testing clients in Moshi, Tanzania, 2003 to 2005.

Antiretroviral treatment literacy leads to greater HIV testing and treatment and antiretroviral treatment adherence. Among northern Tanzanian subjects, antiretroviral treatment awareness was only 17%. Factors associated with low antiretroviral treatment literacy included having exchanged money or gifts for sex, living in rural areas, having more than 2 children, and having a primary education only. Previous HIV testing was protective against low antiretroviral treatment literacy. These results support refocusing HIV education efforts and increasing synergy between HIV prevention and treatment programs.

Capacity of health-care facilities to deliver HIV treatment and care services, Northern Tanzania, 2004.

Few data exist on the current capacity of Tanzanian health-care facilities to deliver antiretroviral therapy (ART). We evaluated this capacity among Northern Zone facilities in 2004 using a questionnaire that addressed human resources, clinical facilities and services, and laboratory capacity. Of 19 facilities surveyed, nine (47%) had staff trained to manage ART and three (16%) prescribed ART. Two (11%) offered CD4 counts, five (26%) offered liver function tests, 16 (84%) offered chest radiography, and 18 (95%) offered acid-fast sputum staining. Of 12 (67%) facilities offering outpatient HIV/AIDS services, 12 (100%) provided co-trimoxazole to outpatients and six (50%) provided isoniazid (INH). All 19 (100%) facilities offered rapid HIV tests and full blood pictures. Overall in 2004, facilities needed strengthening to increase staff training in ART management and to implement INH for treatment of latent tuberculosis. Laboratory facilities for ART monitoring were inadequate, and outpatient ART was limited.

Antigen receptor signaling competence and the determination of B cell fate in B-lymphopoiesis.

Recent studies suggest that developmental check-points in B-lymphopoiesis are set in order to test the B cell receptor signaling competence. In these check-points ligand-independent and ligand-dependent receptor signals confer B-lymphopoiesis with positive and negative selection events. As a consequence, B-lymphocytes are forced to make crucial fate decisions to determine developmental progression, survival or apoptosis. In here we review recent progress in unraveling molecular and cellular mechanisms for the role of B cell receptor signaling competence in determination of the B cell fate.

Cloning and partial characterization of an antigen detected on membrane surfaces of non-pathogenic strains of Entamoeba histolytica.

A novel and unique mAb (318-28) which specifically interacts with a 60 kDa antigen that is found only on the surfaces of non-pathogenic (NP) strains of E. histolytica has been recently characterized. The antigen appears to be present also on (NP) cyst forms of amebas but was not detected on any of the various (P)-strains tested. It was also not found on other Entamoeba species such as Moshkovskii, Laredo, Huff, Coli, Gingivalis, or Invadens. Clinical trails for the differentiation of (P) and (NP) amebas directly from stools using this mAb are in progress. Cloning of the gene encoding for the (NP)-specific antigen was achieved after screening with mAb 318-28 a lambda gt11 expression library of NP strain SAW 1734R. No sequence homology to any known protein was found in data bases.

Cloning and characterization of an unusual elongation factor-1 alpha cDNA from Entamoeba histolytica.

The coding sequence deduced from two overlapping cDNA inserts obtained from a pathogenic strain of Entamoeba histolytica revealed a striking homology (greater than 85%) with elongation factor EF-1 alpha from Saccharomyces cerevisiae and Artemia salina. The deduced amino acid sequence predicted a size of 49 kDa, and antibodies raised against the S. cerevisiae EF-1 alpha cross-reacted with an amoebic protein of similar size (45-47 kDa). Sequence analysis of the cDNA revealed that the 5' untranslated region contained a stretch of 190 nucleotides which was perfectly complementary to a segment of the 3' terminal coding region situated 1015 bases downstream of the methionine initiation codon. Electron microscopy of self-renatured cDNA confirmed the potential of such molecules to form a stem-loop secondary structure. The presence of the complementary sequences was confirmed at the genomic level by sequence analysis of polymerase chain reaction-amplified segments which span both the 3' and 5' terminal complementary regions. Comparison of the deduced amino acid sequence of E. histolytica EF-1 alpha with Ef-Tu from Escherichia coli and EF-1 alpha from different sources, suggested that the major functional domains of the protein are located within the loop structure.

Pathogenic and nonpathogenic strains of Entamoeba histolytica can be differentiated by monoclonal antibodies to the galactose-specific adherence lectin.

Entamoeba histolytica infection results in either asymptomatic colonization or invasive colitis and liver abscess. E. histolytica isolates from patients with invasive disease have characteristic isoenzyme profiles (pathogenic zymodemes), suggesting a role for parasite factors in determining the severity of infection. A galactose-specific cell surface lectin from a pathogenic zymodeme was shown to mediate in vitro adherence to human colonic mucins and contact-dependent killing of target cells. Six nonoverlapping antigenic determinants were identified on the 170-kilodalton heavy subunit of the pathogenic lectin. Anti-lectin monoclonal antibodies (MAb) directed against epitopes 1 and 2 enhanced adherence whereas MAb to epitopes 3 through 6 either inhibited or had no effect on adherence. We tested 50 pathogenic and nonpathogenic strains for reactivity to these anti-lectin MAb by radioimmunoassay. MAb to epitopes 1 through 6 reacted in the radioimmunoassay with all 16 pathogenic zymodeme strains tested. In contrast, only MAb to epitopes 1 and 2 bound to the lectin from nonpathogenic strains. Western immunoblots with anti-lectin antibodies showed that the 170-kilodalton heavy subunit was present in the nonpathogenic amebae. Adherence of the nonpathogenic SAW 760 strain to human erythrocytes was enhanced by MAb to epitope 1 and blocked by galactose, confirming the presence of a functionally active lectin. A lectin radioimmunoassay based on MAb to epitopes 1 and 3 proved to be a simple and rapid method to distinguish pathogenic from nonpathogenic amebae in culture. Further exploration of the functional consequences of the antigenic differences demonstrated for the lectin may lead to a better understanding of its role in pathogenesis.

Low-molecular-weight membrane component inhibits the metastatic phenotype of B16-F10 melanoma.

Treatment of the metastatic melanoma cell lines B16-F1 and B16-F10 with 1.5-2 per cent butanol elevates their experimental metastatic potential, whereas reconstitution of butanol-extracted B16 cells with crude butanol extracts decreases the number of experimentally induced lung foci. We partially purified the biologically active components from crude butanol extracts of B16-F1 by high-performance liquid chromatography and isoelectric focusing, and found the inhibitory activity (i) was in the low-molecular-weight (5-10 kDa) fraction of the chromatogram, (ii) had an isoelectric pH between 5.6 and 5.8, (iii) was distinct from a thiol-protease activity eluted from the isoelectric focusing bed at pH 4.9-5.3 and (iv) was not itself an inhibitor of serine or thiol proteases. Incubation of butanol-extracted B16-F10 cells with known inhibitors of serine, acid and thiol protease inhibitors had no effect on the experimental metastatic phenotype. Although the apparent molecular weight was low, the inhibitor(s) tended to aggregate after focusing, probably owing to the presence of carrier ampholines. Using two-dimensional gel electrophoresis, we observed slight differences between intact and butanol-extracted cells, most of them in the low-molecular-weight region. These results suggest that butanol treatment may reversibly release certain inhibitors of cell surface enzymes other than proteases, which might be involved in invasion and metastasis.

Inhibition of experimental metastasis and extracellular matrix degradation by butanol extracts from B16-F1 murine melanoma.

We previously demonstrated that noncytolytic butanol extraction of B16 melanoma cells can increase the number of experimental lung metastases, and that brief incubation of the extracted cells with the extracted moieties reduces metastatic phenotype. This study examined the possibility that the extracted components are endogenous inhibitors of tumor cell surface-associated, degradative enzymes. The activity was found to be tumor associated, since only tumor extracts could reduce the number of experimental lung metastases of a variety of solid tumors. The activity in crude butanol extracts of B16-F1 that modulated the metastatic phenotype of extracted B16-F10 was partially purified by preparative isoelectric focusing and high-performance gel permeation chromatography. Incubation of extracted B16-F10 cells with low (Mr 2,000-10,000) molecular weight materials focusing in the pH 5.6 to 5.8 region of the preparative isoelectric focusing gradient significantly reduced the number of experimental lung foci. Ampholines alone had no effect. Evidence that the extracted moiety might be an endogenous enzyme inhibitor was obtained with the use of the subendothelial matrix degradation assay, wherein B16-F10 cells digest 35S-labeled heparan sulfate proteoglycan. The same materials that reduced the metastatic potential of butanol-extracted B16-F10 cells also inhibited extracellular matrix degradation by 30 to 85%, as well as the activity of partially purified heparanase (endo-beta-glucuronidase). The metalloproteinase inhibitor 1,10-phenanthroline and the heparanase inhibitor heparin partially (30 to 50%) blocked extracellular matrix degradation. Conversely, inhibitors of serine, thiol, acid, and other proteases had little or no effect on extracellular matrix degradation. These data provide evidence that an endogenous, heat-stable inhibitor of cell surface degradative enzymes such as heparanase may play a role in hematogenous metastasis, and support the hypothesis that butanol extraction activates some of these surface enzymes by removing the endogenous inhibitors.