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Long-term in vitro suspension culture of Toxoplasma and HeLa cells.

To establish a method of mass and long-term propagation of Toxoplasma in suspension cultures and to find a technique for separating the organisms from host cells, basic investigations were carried out in a microcomputer controlled system. By using an arbitrary unit "the medium index" host HeLa cells were continuously supplied to Toxoplasma organisms for about 30 days, at cycles of 4 days. HeLa cells were well infected with Toxoplasma in the suspension cultures by leaving the medium unstirred for 15 min intervals. The separation of Toxoplasma organisms from cells was improved by treating toxoplasmas and HeLa cells mixtures with 0.4% trypsin. The total number of separated organisms obtained from 29 days cultivation period was 4.9 X 10(9). The mean generation time of Toxoplasma organisms on the 0, 10, 20 and 30 day's cultivation was calculated approximately 7.4, 9.4, 9.3 and 7.8 h, respectively.

Animals↗

Comparison of two commercially available avidity tests for toxoplasma-specific IgG antibodies.

BACKGROUND: IgG anti-Toxoplasma avidity tests have proven useful to discriminate between recent and distant infection in management of pregnant women with anti-Toxoplasma-IgM antibodies. This study was performed to compare two commercially available IgG anti-Toxoplasma avidity test kits. METHODS: Sixty-four samples positive for IgM and IgG anti-Toxoplasma antibodies were analyzed with two IgG anti-Toxoplasma avidity tests: VIDAS Toxo IgG avidity kit (BioMérieux, Marcy-l'Etoile, France) and Toxoplasma gondii IgG avidity EIA kit (Labsystems, Helsinki, Finland). Samples were obtained from both pregnant women and subjects in different clinical settings. RESULTS: Of 64 sera positive in both VIDAS Toxo IgG and VIDAS Toxo IgM tests, 19 (29.7%) had high avidity antibodies in VIDAS and 17 (26.6%), high avidity antibodies in Labsystems kit. A total of 48 (75.0%) of 64 sera showed concordant results between VIDAS and Labsystems Toxo IgG avidity tests; one (1.2%) serum sample showed discordant results and in 15 (23.4%) sera, results were high or low in one test and borderline in the other. Overall correlation coefficient for results obtained in both tests was 0.80 (0.60 in sera of pregnant women and 0.88 in sera of other patients, respectively). CONCLUSIONS: Correlation between results obtained in BioMérieux and Labsystems Toxo IgG avidity kits in sera from pregnant women as well other individuals was high. BioMérieux assay possesses the advantage of automatization but requires the VIDAS machine; in contrast, Labsystems assay is less costly but requires time-intensive manual performance and calculation of test results.

Animals↗

Antigens of Toxoplasma gondii recognized by sera of AIDS patients before, during, and after clinically important infections.

A longitudinal study of different parameters of the immune responses to Toxoplasma gondii was performed with sera of AIDS patients taken during and after clinically important Toxoplasma infections. Follow-up of patients lasted for 9 months on an average. The titres of the specific IgG and IgM antibodies were measured by an indirect fluorescent antibody test (IFAT), and the appearance of circulating antigens of Toxoplasma gondii was determined in 88 sera of 18 patients with CNS (6 cases), pulmonary (1), lymph-node toxoplasmosis (1), or asymptomatic primary infections (2), respectively. The profile of the IgG antibodies reacting with a lytic antigen originating from a pool of trophozoites of six different Toxoplasma strains were examined by means of an SDS-PAGE followed by an immunoblot. Although numerous antigen bands were recognized by the sera of patients with clinically important infections, an antigen pattern characteristic of an acute infection could not be discovered. The majority of these sera, however, recognized bands at 27 and 57 kd; proteins of these molecular weights are components of the circulating antigens. In patients without any indication of a Toxoplasma infection, small amounts of antibodies reacting with 34-38 kd antigens were detected. The results of this study demonstrate that seropositivity to Toxoplasma gondii in AIDS patients determined by routine serological methods (e.g. IFAT) may be very heterogeneous even if identical titres are found; it simply results from different combinations of various antibodies which can only be detected by the immunoblotting technique.

Acquired Immunodeficiency Syndrome↗

The role of dopamine in Toxoplasma-induced behavioural alterations in mice: an ethological and ethopharmacological study.

Toxoplasma gondii, a cosmopolitan protozoan parasite, is known to induce behavioural alterations in rodents and may exert an effect on human personality and behaviour. The mechanism of parasite-induced alterations in host behaviour has not been described, but it was hypothesized that development of Toxoplasma tissue cysts in the brain could affect the dopaminergic neuromodulatory system. In this study, we tested the effect of latent Toxoplasma infection on mouse behaviour associated with activity of the dopaminergic system, i.e. locomotion in a novel environment and exploration test. Additionally, we examined the behavioural response of Toxoplasma-infected mice to a selective dopamine uptake inhibitor, GBR 12909. In both genders, Toxoplasma infection decreased locomotion in the open field. Infected females displayed an increased level of exploration in the holeboard test. GBR 12909 induced suppression in holeboard-exploration in the infected males, but had an opposite effect on the controls. These results suggest an association between Toxoplasma gondii infection and changes in the dopaminergic neuromodulatory system.

Animals↗

SPECT thallium-201 combined with Toxoplasma serology for the presumptive diagnosis of focal central nervous system mass lesions in patients with AIDS.

OBJECTIVE: To determine the utility of brain thallium-201 single photon emission computerized tomography (Tl-201 SPECT) combined with Toxoplasma serology for the diagnosis of focal CNS lesions in patients with AIDS. METHODS: Sixty-one consecutive HIV-infected patients with focal CNS lesion(s) on head computed tomography (CT) or MRI scan who underwent brain Tl-201 SPECT and serum Toxoplasma serology were evaluated, retrospectively. Thallium-201 uptake ratios were calculated by comparing lesion activity to contralateral scalp activity. Diagnoses were made by a combination of histology, serology, PCR, and empirical response to therapy. Toxoplasma serologies (IgG IFA) were compared in the patients with central nervous system (CNS) toxoplasmosis and those without CNS toxoplasmosis. RESULTS: Fifty-six patients were evaluable and a definitive diagnosis was made in 38 patients: toxoplasmosis (17), lymphoma (14), PML (three), Aspergillus (one), tuberculoma (one), Cryptococcus (one), varicella-zoster virus (one). Patients with lymphoma had significantly higher lesion/contralateral scalp ratios compared to patients without lymphoma: 1.03 vs. 0.67, P < 0.05. Using a cut-off of 0.90 for the lesion/scalp uptake ratios (based on analysis of ROC curves) the sensitivity and specificity for the diagnosis of lymphoma were 86% and 83%, respectively. Serum Toxoplasma IgG titres were significantly higher in patients diagnosed with toxoplasmosis compared to those with a diagnosis other than toxoplasmosis, 1:5444 vs. 1:15, P < 0.05. Only one patient with confirmed toxoplasmosis had a Toxoplasma serology < 1:256, while no patients without toxoplasmosis (including all lymphoma patients) had serologies > 1:256. CONCLUSIONS: In a series of HIV-infected patients, Tl-201 SPECT was able to accurately differentiate primary brain lymphoma from other causes of focal CNS lesions in most patients; however, both false positive and false negative results occurred. By combining Tl-201 SPECT with serum Toxoplasma IgG, diagnostic accuracy was improved.

AIDS-Related Opportunistic Infections↗

Confirmatory serologic testing for acute toxoplasmosis and rate of induced abortions among women reported to have positive Toxoplasma immunoglobulin M antibody titers.

OBJECTIVE: Results obtained with commercial testing kits for immunoglobulin M Toxoplasma antibodies may be inaccurate or may be inaccurately interpreted, which may influence whether a woman decides to terminate the pregnancy. This study was undertaken to determine whether confirmatory testing at a reference laboratory and communication of the results and an expert interpretation to the patient's physician would affect the rate of induced abortions among pregnant women with positive results of testing for immunoglobulin M Toxoplasma antibodies in outside laboratories. STUDY DESIGN: This was a retrospective cohort study of 811 consecutive pregnant women for whom the toxoplasma serologic profile was performed at a reference laboratory. Almost all the patients had been informed by their physicians that a result of a test for immunoglobulin M Toxoplasma antibodies performed in an outside laboratory was positive. Women were separated into those with a toxoplasma serologic profile result suggestive of a recently acquired infection (group 1) and those with a result suggestive of an infection acquired in the more distant past (group 2). Physician reports of induced abortions were used to determine rates of induced abortion in groups 1 and 2. RESULTS: Of the 811 women 321 (39.6%) were considered likely to have a recent infection (group 1) and 490 (60.4%) were considered likely to have a past infection (group 2). Physicians reported pregnancy outcomes for 433 (53.4%) of 811 women (65.1% and 45.7% in groups 1 and 2, respectively). Whereas 36 of 209 women in group 1 (17.2%) terminated the pregnancy, only 1 of 224 women in group 2 (0.4%) chose abortion (P <.001). CONCLUSION: Confirmatory serologic testing in a reference laboratory and communication of the results and their correct interpretation by an expert to the patient's physician decreased the rate of unnecessary abortions by approximately 50% among women for whom positive immunoglobulin M Toxoplasma test results had been reported by outside laboratories.

Abortion, Induced↗

T cell blastogenic responses to Toxoplasma gondii trophozoites among HIV-infected patients.

OBJECTIVE: To evaluate the cell-mediated immune response to Toxoplasma gondii (T. gondii) among HIV-infected patients. METHODS: Forty HIV-infected patients were studied. Of them, 35 had antibodies to T. gondii and 5 had not. Nine of 35 patients with detectable serum Toxoplasma antibodies were classified as group A1 (CDC 1993), 10 as group B2, 6 as group B3, and 10 as group C3. Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque gradient centrifugation. Living Toxoplasma gondii trophozoites, herpes simplex virus (HSV), tetanus toxoid, and phytohemoagglutinin (PHA) were used in standard proliferation assays. Toxoplasma-responding blasts were expanded and assayed for antigen specificity and HLA restriction by proliferation assays. T cell subsets were analyzed using two-color flow cytometry. RESULTS: Among patients with detectable Toxoplasma serum antibodies, significant PBMC proliferation in response to T. gondii trophozoites was observed in those classified in group A1 or B2 but not in those in groups B3 and C3. Toxoplasma-induced blasts from five of six patients after 7 days of culture and from five patients after 15 days of culture proliferated in response to T. gondii in the presence of either autologous or allogeneic PBMCs as antigen-presenting cells (APCs) and/or also proliferated in response to HSV. The surface markers of T. gondii-induced blasts showed a variable percentage of CD4 and CD8 activated cells. CONCLUSIONS: T cell proliferative response to living trophozoites of T. gondii is lost only in patients with severe depletion of CD4 cells. PBMC proliferation was observed only in patients with previous T. gondii infection, but the T cell blasts generated showed a strong alloreactivity (proliferating in response to allogeneic irradiated PBMCs) and were apparently not antigen specific (proliferating also in response to HSV).

Adult↗

Recent primary toxoplasma infection indicated by a low avidity of specific IgG.

An unusual assay was developed for the serologic diagnosis of recent primary infection by Toxoplasma gondii. This test measures the antigen-binding avidity of toxoplasma-specific IgG antibodies. Serum samples from 5 patients with recent primary toxoplasma infection were compared with those from 21 subjects with preexisting toxoplasma immunity. Patients with primary infection exhibited a low avidity of toxoplasma-specific IgG, which persisted for several months after the onset of symptoms of toxoplasmosis. In contrast, all subjects with past immunity had a high avidity of toxoplasma-IgG. This IgG avidity assay should assist in the diagnosis of acquired toxoplasmosis and may be used to identify pregnancies that are at risk for congenital toxoplasmosis.

Adult↗

Animal exposures and antibodies to Toxoplasma gondii in a university population.

To determine the risk of toxoplasma infection to individuals exposed to cats in a research institution, we compared the prevalence of toxoplasma antibodies with exposure to cats in university employees. Of 116 employees tested, 42 (36 percent) had toxoplasma antibodies as determined by the indirect fluorescent antibody test. Women and individuals aged 35 years or more had a greater prevalence of antibodies. The antibody prevalence by occupation was 72.1 percent for physicians and those with doctorates, 45.3 percent for animal and veterinary technicians, 33.3 percent for research technicians, 28.2 percent for administrative staff, 25.0 percent for graduate students and fellows, and 13.4 percent for veterinarians. There was no significant positive association between exposure to cats and the prevalence of toxoplasma antibodies. A follow-up of seronegative employees, 6 and 18 months later, revealed no seroconversions indicative of acute toxoplasma infection. We concluded that there was no significant risk of toxoplasma infection in university employees exposed to cats.

Adult↗

The host cell transcription factor hypoxia-inducible factor 1 is required for Toxoplasma gondii growth and survival at physiological oxygen levels.

Toxoplasma gondii is an obligate intracellular protozoan pathogen. We previously found that genes mediating cellular responses to hypoxia were upregulated in Toxoplasma -infected cells but not in cells infected with another intracellular pathogen, Trypanosoma cruzi. The inducible expression of these genes is controlled by the hypoxia-inducible factor 1 (HIF1) transcription factor, which is the master regulator of cells exposed to low oxygen. Because this response may be important for parasites to grow at physiological oxygen levels, we tested the hypothesis that HIF1 is important for Toxoplasma growth. Here, we demonstrate that Toxoplasma infection rapidly increased the abundance of the HIF1alpha subunit and activated HIF1 reporter gene expression. In addition, we found that Toxoplasma growth and survival was severely reduced in HIF1alpha knockout cells at 3% oxygen. While HIF1alpha was not required for parasite invasion, we determined that HIF1 was required for parasite cell division and organelle maintenance at 3% oxygen. These data indicate that Toxoplasma activates HIF1 and requires HIF1 for growth and survival at physiologically relevant oxygen levels.

Animals↗

Seroprevalence of Toxoplasma gondii in northern Greece during the last 20 years.

The seroprevalence of Toxoplasma gondii in the northern Greek population was determined in 1984, 1994 and 2004, and changes during this period were investigated. In total, 1014, 812 and 958 sera from individuals aged 1 day to 70 years were examined in 1984, 1994 and 2004, respectively, for IgG and IgM anti-Toxoplasma antibodies with the standard immunofluorescence assay (IFA) and microparticle enzyme immunoassay (MEIA). In individuals positive for IgM-specific antibodies, primary infection with Toxoplasma was diagnosed on the basis of the Toxoplasma serological profile (IFA, MEIA, conventional IgM and IgA ELISAs, immunosorbent agglutination assay and IgG avidity test). The prevalence of IgG-specific antibodies in the general population was 37%, 29.9% and 24.1% in 1984, 1994 and 2004, respectively, and was 35.6%, 25.6% and 20%, respectively, in women of reproductive age (15-39 years). The incidence of Toxoplasma infection, based on cases of primary infection and the annual seroconversion rate for the general population, was estimated to be 1.25% and 1.1% in 1984, 1.05% and 0.93% in 1994, and 0.85% and 0.8% in 2004. The significant decline in prevalence, and the shift towards an older age group, observed during this period could be explained by the improved socio-economic situation. The high (80%) proportion of women of reproductive age susceptible to Toxoplasma infection, with an estimated 90-200 neonates infected in utero annually, seems to present a potential risk to public health. Education of the public and prophylactic measures may become increasingly important.

Adolescent↗

Scanning electron microscopy of Toxoplasma gondii: parasite torsion and host-cell responses during invasion.

Scanning electron microscopy confirmed our previous finding that toxoplasmas actively invade mouse peritoneal cells that are inhibited from phagocytosis. The parasites entered cells with the conoid end first and sometimes showed a counter-clockwise torsion of the body during invasion. Counter-clockwise torsion was also noted in free toxoplasmas. Host-cell responses to active invasion varied with experimental conditions and with the type of host cell. Under adverse culture conditions for phagocytosis, normal macrophages formed rudimentary filopodia or lamellipodia around the tips of invading toxoplasmas; macrophages subjected to hyperthermia before similar incubation with toxoplasmas showed little or no response to invasion. Normal and heat-treated lymphocytes showed little surface reaction to invasion, but occasionally a flocculent collar was seen around the tip of an invading toxoplasma. Scanning electron microscopy provides clues to possible mechanisms of toxoplasma locomotion and host-cell invasion.

Animals↗

Control of congenital infection with Toxoplasma gondii by neonatal screening based on detection of specific immunoglobulin M antibodies eluted from phenylketonuria filter-paper blood-spot samples.

Two ongoing neonatal screening programmes for congenital infection with Toxoplasma gondii are presented. The New England Newborn Screening Programme has included congenital toxoplasmosis since 1986. The test is based on detection of Toxoplasma-specific immunoglobulin M (IgM) antibodies eluted from the phenylketonuria (PKU) card. The seroprevalence of Toxoplasma IgG antibodies is at present about 13% and the birth prevalence of congenital toxoplasmosis approximately 1 per 10000 liveborn children. The Danish national neonatal screening programme was expanded to include congenital toxoplasmosis from 1 January 1999. The test is also based on detection of Toxoplasma-specific IgM antibodies eluted from PKU cards. The seroprevalence of Toxoplasma IgG antibodies in pregnant women is around 25% and the birth prevalence about 1 per 3000 liveborn children. The birth prevalence of congenital Toxoplasma infection is within the range of other congenital disorders included in different screening programmes. Neonatal screening is feasible in areas with a low risk of congenital infection where prenatal screening will not be applicable.

Animals↗

False-positive anti-Toxoplasma fluorescent-antibody tests in patients with antinuclear antibodies.

The indirect fluorescent-antibody (IFA) method for diagnosis of toxoplasmosis is widely used and is considered to be as specific as the Sabin-Feldman dye test. After observing a patient with systemic lupus erythematosus (SLE) who had a positive toxoplasma IFA test but a negative dye test, we studied sera with high titers of antinuclear antibodies from 16 SLE patients and from 2 with rheumatoid arthritis for Toxoplasma antibodies in the immunoglobulin G and M (IgG and IgM) IFA tests and the dye test. Results of these tests were compared with titers of antinuclear antibodies, precipitating antibodies to single-strand deoxyribonucleic acid (DNA), and binding antibodies by use of DNA labeled with (3)H-actinomycin D. Of 18 patients, 11 had IgG and 4 had IgM IFA Toxoplasma antibodies; only 2 had antibodies detectable in the dye test. The immunofluorescence patterns in the Toxoplasma IFA test were indistinguishable from those obtained in patients with toxoplasmosis without antinuclear antibodies. Absorption of SLE sera with DNA did not result in a decrease in Toxoplasma IFA titers. When SLE sera were absorbed with live T. gondii, a marked drop in IgG IFA titer was observed as well as a decrease in titers of antinuclear antibodies and (3)H-DNA binding. Treatment of Toxoplasma cells with deoxyribonuclease and ribonuclease did not decrease their fluorescence. These results suggest that T. gondii nuclear antigens can absorb antinuclear antibodies but do not have exposed substrates for deoxyribonuclease. Tests in which organisms containing "nuclear" antigens for IFA detection of antibodies to these organisms are used may result in "false-positives" with sera containing antinuclear antibodies.

Absorption↗

Cross-immunity between Hammondia and Toxoplasma infections in mice and hamsters.

Cross-immunity between six strains of Hammondia hammondi and the M-7741 strain of Toxoplasma gondii was studied in mice and hamsters. Mice and hamsters were inoculated orally with 10(5) H. hammondi oocysts. All mice and hamsters survived. Four weeks later, they were challenged with 1 to 10(5) mean lethal doses (LD50) of the Toxoplasma oocysts. Animals that died were necropsied. Survivors were killed 30 days after challenge inoculation, and the number of cysts in brain and musculature was determined and compared with that of animals that were not immunized with H. hammondi. In one experiment, 36 mice were immunized with each of three H. hammondi strains. Four weeks later the mice were challenged with 1 to 10(5) LD50 doses of T. gondii oocysts. Of 108 mice immunized with H. hammondi, 103 survived challenge with Toxoplasma oocysts for 30 days, whereas only 3 of 36 unimmunized mice survived with similar doses of Toxoplasma oocysts. Fewer Toxoplasma cysts were found in mice immunized with H. hammondi than in unimmunized mice. In another experiment groups of six hamsters were each immunized with one of six H. hammondi strains and then challenged with 10(5) LD50 Toxoplasma oocysts. All unimmunized hamsters died between 9 and 13 days after inoculation. Percent protection in the various groups of immunized hamsters was: 100, 84, 66, 66, 50, and 33.

Animals↗

Effect of alveolar macrophages on Toxoplasma gondii.

As pulmonary involvement can occur in disseminated toxoplasmosis in immunosuppressed patients, studies were initiated to define local mechanisms of resistance of the lung to Toxoplasma gondii. Alveolar macrophages were obtained from normal mice and mice chronically infected with T. gondii by bronchopulmonary lavage and cultured in vitro. Although normal alveolar macrophages were difficult to infect with Toxoplasma, they supported intracellular multiplication of this organism. When exposed to Toxoplasma that had been pretreated with heat-inactivated serum containing specific antibody, the number of intracellular organisms increased remarkably, and the macrophages destroyed the coated parasites. After development of chronic infections with Toxoplasma, there was a transient period during which a striking increase in numbers of alveolar macrophages was observed in lavage specimens. These macrophages differed from those of normal alveolar macrophages. There was a greater percentage of large cells, a greater tendency to spread on glass, and an increased number of intracellular Toxoplasma, and the cells were activated to kill or inhibit multiplication of the parasite. During the period when activated macrophages were demonstrable in bronchopulmonary washings, histological changes in the lungs revealed a marked mononuclear cell infiltrate. These studies support a role for the activated alveolar macrophage as an effector in resistance of the lung to infection with Toxoplasma.

Animals↗

Regulation of immunoglobulin E production in mice immunized with an extract of Toxoplasma gondii.

Repeated infection with Toxoplasma gondii could not induce immunoglobulin E (IgE) antibody production. When mice were injected intraperitoneally twice over a 3-week interval with an extract of tachyzoites of T. gondii and Al(OH)3 as adjuvant, antitoxoplasma IgE antibody was produced. Antitoxoplasma IgE antibody titers were low and diminished after a short time in B10.S, BALB/c, C3H, and C57BL/6 mice. This tendency was more evident in IgE low-responder SJL mice. IgE-inducing activity of Toxoplasma antigen was weaker than those of keyhole limpet hemocyanin, ovalbumin, and an extract from Nippostrongylus brasiliensis. The antitoxoplasma IgE antibody production was enhanced by and persisted after whole-body irradiation (150 R) following secondary immunization. The enhanced antitoxoplasma IgE antibody production was suppressed by transferring spleen cells from Toxoplasma antigen-immunized mice. The suppressive effect of the spleen cells was Toxoplasma antigen specific and was removed by treatment with anti-Thy-1.2 and complement. These results indicate that the low IgE production induced by Toxoplasma antigen is the result of irradiation-sensitive and antigen-specific suppressor T cells. These findings might explain the lack of IgE antibody response in mice with Toxoplasma infection.

Animals↗

European multicenter study of the LIAISON automated diagnostic system for determination of Toxoplasma gondii-specific immunoglobulin G (IgG) and IgM and the IgG avidity index.

The LIAISON system is a fully automated system based on chemiluminescence and antigen bound to magnetic microparticles. The system allows fast and precise measurement of Toxoplasma-specific immunoglobulin G (IgG) and IgM antibody levels and measurement of the IgG avidity index even at low levels of Toxoplasma-specific IgG antibodies in a single step without manual interference. Seven European centers participated in a multicenter evaluation of the LIAISON system. The sensitivity and specificity of the LIAISON system compared to the Sabin-Feldman dye test were 99.3 and 96.8%, respectively. In a comparison of the LIAISON Toxoplasma-specific IgM assay with an immunosorbent agglutination assay, the LIAISON assay had a sensitivity of 96.7% and a specificity of 95.4%. The LIAISON IgG assay showed agreements of 91, 100, and 100% with the AXSYM IgG (Abbott), VIDAS IgG (bioMerieux), and Platelia IgG (Bio-Rad) assays, respectively. The LIAISON IgM assay showed agreements of 95% with the AXSYM IgM and Platelia IgM assays, 96% with the ISAGA IgM assay (bioMerieux), and 97% with the VIDAS IgM assay. The coefficient of correlation between the LIAISON system and the VIDAS Toxoplasma-specific IgG avidity index was 0.81. By use of the Toxoplasma-specific IgG avidity index assay with specific IgM-positive samples, the diagnosis of infection with Toxoplasma gondii in early pregnancy has been improved significantly. The LIAISON avidity assay is a valuable assay for the exclusion of recently acquired infection with T. gondii (less than 4 months) in pregnant women, and it decreases significantly the necessity for follow-up testing.

Animals↗