Toxoplasmic encephalitis in AIDS-patients before and after the introduction of highly active antiretroviral therapy (HAART).

Toxoplasmic encephalitis (TE) continues to be a severe health problem despite the introduction of highly active antiretroviral therapy (HAART). To identify predictors for development of TE we compared demographic, clinical and diagnostic variables in AIDS patients with TE before (n = 102) or after the introduction (n = 70) of HAART at the Charité University Medicine in Berlin, Germany. Interestingly, patient characteristics did not differ significantly in the pre- and post-HAART groups. Sixty-eight percent of patients had CD4-cell counts of <50/µl. Outcome after treatment with pyrimethamin plus sulfonamides or clindamycin (47% each) did not differ; adverse reactions were more frequent in patients receiving sulfonamides than in those receiving clindamycin (25% vs. 10.5%; p = 0.02). Interestingly, patients in the post HAART group had not received (82.9%) or had not taken HAART adequately (17.1%). Concurrent diagnosis of TE and HIV was significantly more often in the post- compared to the pre-HAART group (49 vs. 26%, respectively; p > 0.001). Thus, despite the introduction of HAART, awareness of opportunistic infections in HIV patients is warranted. High rates of unawareness of HIV infection should make public health efforts focus on early identification of HIV infection and initiation of and compliance with HAART.

Toxoplasma gondii infection in workers occupationally exposed to raw meat.

BACKGROUND: Raw meat may contain viable Toxoplasma gondii tissue cysts and therefore handling of raw meat may represent a risk for T. gondii infection. AIMS: To determine the association of T. gondii infection with occupational exposure to raw meat. METHODS: Case-control seroprevalence study design with enzyme-linked immunoassays for the presence and levels of anti-Toxoplasma IgG antibodies and for the presence of anti-Toxoplasma IgM antibodies. Those occupationally exposed to raw meat consisted of butchers working in two abattoirs and 35 butcher's shops in Durango, Mexico. The control group consisted of individuals from the general population from the same region. Socio-demographic, work, clinical and behavioural characteristics from each butcher were obtained. RESULTS: One hundred and twenty-four workers occupationally exposed to raw meat and 248 control subjects were examined. Eight (7%) of the butchers and 22 (9%) of the controls were positive for anti-T. gondii IgG antibodies [not statistically significant (NS)]. Anti-T. gondii IgG levels were >150 IU/ml in 7 (6%) butchers and 14 (6%) controls (NS). Anti-T. gondii IgM antibodies were found in five (4%) of the butchers and four (2%) of the controls (NS). None of the factors examined appeared to predict seropositivity although both butchers who reported consuming dried beef were seropositive compared to 6/122 controls (95% CI 0.60-1.29). CONCLUSIONS: Occupational exposure to raw meat was not associated with seropositivity for T. gondii infection. Consumption of dried beef may warrant further investigation.

Association of CD4+ T cell-dependent, interferon-gamma-mediated necrosis of the small intestine with genetic susceptibility of mice to peroral infection with Toxoplasma gondii.

Since there is a remarkable difference in susceptibility to peroral infection with Toxoplasma gondii among inbred strains of mice, we performed studies to examine the mechanism(s) of this difference in susceptibility. After peroral infection with the ME49 strain of T. gondii, C57BL/6 (B6) mice all died whereas BALB/c mice all survived. At day 7 of infection (when B6 mice began dying), massive necrosis of the villi and mucosal cells in the ilea were observed in B6 but not in BALB/c mice. To analyze the role of T cells in resistance against death and development of necrosis in the ilea after infection, studies were performed using athymic nude and euthymic control B6 and BALB/c mice. Athymic B6 mice all died after infection, but surprisingly, they survived significantly longer than control B6 mice, indicating that T cells predispose to early death in these mice. Necrosis in the ilea was observed in control B6 but not in athymic B6 mice; however, significantly less numbers of tachyzoites were observed in the ilea of the former than the latter mice. These results indicate that necrosis in the ilea of the B6 mice was not due to destruction of tissue by tachyzoites but was mediated by T cells. This deleterious effect of T cells appears to contribute to early death in these mice. In contrast, T cells conferred resistance against death in BALB/c mice but did not cause necrosis in their ilea. To analyze the T cell subset(s) that induces necrosis of the ilea in B6 mice, we examined histological changes of the small intestines after infection of mutant mice deficient in different T cell subsets (with the same H-2b haplotype as B6 mice). Mice deficient in alpha/beta or CD4+ T cells did not develop necrosis in the ilea, whereas wild-type control mice and mice deficient in gamma/delta or CD8+ T cells did, suggesting that the cells that induce necrosis in the ilea after infection are CD4+ alpha/beta T cells. Since interferon (IFN)-gamma has been shown to be critical for survival of BALB/c mice after infection with T. gondii, we examined the role of this cytokine in resistance/susceptibility of infected B6 mice. Treatment of B6 mice with anti-IFN-gamma monoclonal antibody shortly before they developed illness prolonged time to death and prevented necrosis in the ilea in these mice. These results indicate that IFN-gamma mediates necrosis in the ilea of B6 mice after infection. This CD4+ T cell-dependent, IFN-gamma-mediated necrosis of the small intestines appears to be a mechanism that underlies the genetic susceptibility of B6 mice to peroral infection with T. gondii, whereas the same cytokine plays a critical role in the resistance of genetically resistant BALB/c mice.

99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: induction and control of regulatory T cells in the gastrointestinal tract: consequences for local and peripheral immune responses.

Regulatory T cells play a crucial role in normal gut homeostasis, as well as during infection with microbial or parasitic pathogens. Prior to infection, interactions with the commensal microflora are essential to differentiation of a healthy steady-state level of immunoregulation, mediated through both Toll-like receptor-dependent and -independent pathways. The ingress of pathogenic organisms may, according to the context, promote or reverse the regulatory environment, with onward consequences for inflammation in both the intestinal and extra-intestinal settings. Appropriate regulation of gut immunity thus depends upon a complex three-way interplay between host cells, commensals and pathogens, and can exert a major impact on systemic responses including allergy and autoimmunity.

Neutralization assay to resolve discrepancies between positive results in new highly sensitive anti-Toxoplasma gondii IgG assays and negative results in reference tests.

High sensitivity of IgG assays is required in order to diagnose Toxoplasma infection in immunocompromised patients and women before and during early pregnancy. Positive results obtained in new highly sensitive Toxoplasma IgG assays have been observed in sera that gave negative results in reference assays. To allow the resolution of these discrepancies, we developed a neutralization assay using 80 microg/ml of a soluble membrane fraction of Toxoplasma in the automated Elecsys IgG assay. We were able to block the detection of Toxoplasma IgG antibodies in 283 sera with IgG titers ranging from below the limit of detection to >2,300 IU/ml. The mean percentage of neutralization was 88%, with a minimum neutralization of 77%. In summary, the neutralization assay represents a valuable tool to confirm the specificity of Toxoplasma IgG antibodies in the sera of immunocompromised patients and women before and during early pregnancy. Discrepancies between positive results in highly sensitive Toxoplasma IgG assays and negative results in reference assays can, thereby, be resolved and may allow a decision to be made on the management of patients.

Congenital infections with Trypanosoma cruzi or Toxoplasma gondii are associated with decreased serum concentrations of interferon-γ and interleukin-18 but increased concentrations of interleukin-10.

Little is known about the immune responses of newborns with congenital Chagas disease (CCD) or congenital toxoplasmosis (CT) but they probably differ to those seen in adults with Chagas disease or toxoplasmosis, leading to differences in pathology. The concentrations of interleukin-18 (IL-18), interferon-γ (IFN-γ) and interleukin 10 (IL-10) in the sera of infants with CCD or CT were determined and compared with those in the sera of uninfected controls (born to mothers who were seropositive or seronegative for Trypanosoma cruzi). The infants with CCD or CT were found to have lower IL-18 and IFN-γ concentrations but higher IL-10 concentrations than the uninfected controls. The IL-18 and IFN-γ concentrations were also significantly lower in the infants with CCD than in those with CT. Although the infants with symptomatic CT had significantly higher serum concentrations of IL-18 than those with asymptomatic infection with Toxoplasma, the infants with symptomatic CCD had similar serum concentrations of IL-18 to the infants with asymptomatic Tr. cruzi infection. Taken together, these results indicate that IL-10 contributes to the suppression of pro-inflammatory immune responses and therefore, perhaps, to clinically overt CCD and CT.

Seroepidemiology of Toxoplasma gondii infection in pregnant women in rural Durango, Mexico.

The epidemiology of Toxoplasma gondii infection in pregnant women in rural Mexico is largely unknown. The seroepidemiology of T. gondii infection in 439 pregnant women from 9 communities in rural Durango State, Mexico was investigated. Using commercial enzyme-linked immunoassays, sera were tested for T. gondii IgG, IgM, and avidity antibodies. Prevalences of T. gondii IgG antibodies in the communities varied from 0% to 20%. Overall, 36 (8.2%) of the 439 women had IgG T. gondii antibodies. Ten (2.3%) women had also T. gondii IgM antibodies; IgG avidity was high in all IgM-positive women, suggesting chronic infection. None of the women, however, had delivered a known T. gondii-infected child. The seroprevalence was significantly higher (P < 0.05) in women from low socio-economic conditions (14%) than in those with higher socio-economic status (6.6%). Multivariate analysis showed that T. gondii infection was associated with soil floors at home (adjusted OR = 2.89; 95% CI: 1.12-7.49). This is the first epidemiological study of T. gondii infection in pregnant women in rural Mexico.

Liquid and Dry Swabs for Culture- and PCR-Based Detection of Colonization with Methicillin-Resistant Staphylococcus aureus during Admission Screening.

Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) colonization status facilitates isolation and decolonization and reduces MRSA infections. Liquid but not dry swabs allow fully automated detection methods. However, the accuracy of culture and polymerase chain reaction (PCR) using liquid and dry swabs has not been analyzed. We compared different swab collection systems for routine nasal-throat MRSA screening in patients admitted to a tertiary care trauma center in Germany. Over 3 consecutive months, dry swabs (month 1), ESwabs (month 2), or MSwabs (month 3) were processed using Cepheid GeneXpert, Roche cobas and BD-MAX™ MRSA tests compared to chromogenic culture. Among 1680 subjects, the MRSA detection rate using PCR methods did not differ significantly between dry swabs, ESwab, and MSwab (6.0%, 6.2%, and 5.3%, respectively). Detection rates using chromogenic culture were 2.9%, 3.9%, and 1.9%, using dry, ESwab, and MSwab, respectively. Using chromogenic culture as the "gold standard", negative predictive values for the PCR tests ranged from 99.2-100%, and positive predictive values from 33.3-54.8%. Thus, efficient and accurate MRSA screening can be achieved using dry, as well as liquid E- or MSwab, collection systems. Specimen collection using ESwab or MSwab facilitates efficient processing for chromogenic culture in full laboratory automation while also allowing molecular testing in automated PCR systems.

Seroepidemiology of Toxoplasma gondii infection in human adults from three rural communities in Durango State, Mexico.

There is scarce information concerning the epidemiology of Toxoplasma gondii infection in people of rural Mexico. Anti-T. gondii immunoglobulin (Ig)G and IgM antibodies were sought in 462 adult inhabitants from 3 rural communities of Durango, Mexico, using enzyme-linked immunoassays. In total, 110 (23.8%) of 463 persons had IgG anti-T. gondii antibodies. Ten (2.2%) of them also had IgM anti-T. gondii antibodies. Prevalences of T. gondii IgG antibodies in the 3 communities varied from 14.8 to 35.8%. The highest prevalence of infection was observed in participants older than 70 yr and in those with good housing conditions. Toxoplasma gondii infection was significantly associated with consumption of squirrel (adjusted odds ratio [OR] = 4.22; 95% confidence interval [CI] = 1.11-16.05) and turkey meat (adjusted OR = 4.58; 95% CI = 1.14-18.44). This is the first epidemiological study of T. gondii prevalence in rural Mexico.

Targeted inactivation of the tetraspanin CD37 impairs T-cell-dependent B-cell response under suboptimal costimulatory conditions.

CD37 is a membrane protein of the tetraspanin superfamily, which includes CD9, CD53, CD63, CD81, and CD82. Many of these molecules are expressed on leukocytes and have been implicated in signal transduction, cell-cell interactions, and cellular activation and development. We generated and analyzed mice deficient for CD37. Despite the high expression of CD37 on cells of the immune system, no changes in development and cellular composition of lymphoid organs were observed in mice lacking CD37. Analyses of humoral immune responses revealed a reduced level of immunoglobulin G1 (IgG1) in the sera of nonimmunized mice and an alteration of responses to T-cell-dependent antigens. Antibody responses to model antigen administered in the absence of adjuvant and to viral infections were generally poor in CD37-deficient mice. These poor antibody responses could be overcome by the immunization of antigen together with adjuvant. These results suggest a role for CD37 in T-cell-B-cell interactions which manifests itself under suboptimal costimulatory conditions.

Seroprevalence of Toxoplasma gondii antibodies in cats from Durango City, Mexico.

The prevalence of antibodies to Toxoplasma gondii was determined in sera from 105 domestic cats from Durango City, Mexico. Using a modified agglutination test, antibodies to this parasite were found in 21% of the 105 cats, with titers of 1:25 in 3 cats, 1:50 in 4 cats, 1:200 in 5 cats, 1:400 in 2 cats, 1:800 in 2 cats, 1:1,600 in 4 cats, and 1:3,200 or higher in 2 cats. Cats older than 1 yr had a significantly higher frequency of infection than that found in cats younger than 0.5 yr (41 vs. 13.2%, respectively; odds ratio = 4.55; 95% CI = 1.24-17.18; P = 0.01). Overall, the seroprevalence of T. gondii antibodies in cats in Durango, Mexico, is much lower compared with those reported in other countries.

Neospora caninum and Toxoplasma gondii antibodies in dogs from Durango City, Mexico.

Toxoplasma gondii and Neospora caninum are structurally similar parasites, with many hosts in common. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs from Durango City, Mexico. Using a modified agglutination test, antibodies to T. gondii were found in 52 (51.5%) of the 101 dogs with titers of 1:25 in 27, 1:50 in 11, 1:100 in 5, 1:200 in 4, 1:400 in 2, 1:800 in 2, and 1:3,200 or higher in 1. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT) and the Neospora sp. agglutination test (NAT). Two of the 101 dogs had N. caninum antibodies; these dogs did not have T. gondii antibodies, supporting the specificity of the tests used. The N. caninum antibody titers of the 2 dogs were: 1:400 by IFAT and 1:200 by NAT in 1, and 1:25 by NAT and IFAT in the other. Results indicate that these 2 structurally similar protozoans are antigenically different.

[Ocular toxoplasmosis]. / Okuläre Toxoplasmose.

Toxoplasmosis is a parasitic zoonosis which occurs worldwide and is an important cause of blindness. The infection is naturally acquired by the ingestion of oocysts excreted by infected cats or by ingestion of tissue cysts in undercooked or raw meat. Primary infection during pregnancy may result in a congenital infection. Toxoplasmic retinochoroiditis is the most common cause of posterior uveitis in immunocompetent patients. Depending on the patient's age, ocular symptoms vary presenting with reduced visual acuity, strabismus, and nystagmus in young children - in adults decreased vision and floaters are most frequently reported. Active toxoplasmic retinochoroiditis typically presents as grey-white retinal necrosis with choroiditis, vasculitis and vitritis. However, atypical presentations including neuroretinitis, papillitis, Fuchs-like anterior uveitis, scleritis and acute retinal necrosis have been described. The diagnosis is based on clinical findings and can be supported by the detection of antibodies and Toxoplasma gondii DNA. Toxoplasmosis therapy includes antimicrobial drugs and corticosteroids. There are several regimens with different drug combinations including, among others, pyrimethamine, sulfadiazine, clindamycin, and trimethoprim-sulfamethoxazol. The prognosis for ocular toxoplasmosis is favorable in immunocompetent individuals, as long as the central macula is not directly involved. The present article reviews the epidemiology, pathogenesis, clinical presentation and management of toxoplasmic retinochoroiditis.

Toxoplasmosis.

Toxoplasma gondii is a protozoan parasite that infects up to a third of the world's population. Infection is mainly acquired by ingestion of food or water that is contaminated with oocysts shed by cats or by eating undercooked or raw meat containing tissue cysts. Primary infection is usually subclinical but in some patients cervical lymphadenopathy or ocular disease can be present. Infection acquired during pregnancy may cause severe damage to the fetus. In immunocompromised patients, reactivation of latent disease can cause life-threatening encephalitis. Diagnosis of toxoplasmosis can be established by direct detection of the parasite or by serological techniques. The most commonly used therapeutic regimen, and probably the most effective, is the combination of pyrimethamine with sulfadiazine and folinic acid. This Seminar provides an overview and update on management of patients with acute infection, pregnant women who acquire infection during gestation, fetuses or infants who are congenitally infected, those with ocular disease, and immunocompromised individuals. Controversy about the effectiveness of primary and secondary prevention in pregnant women is discussed. Important topics of current and future research are presented.

Performance of the Cobas(®) Influenza A/B Assay for Rapid Pcr-Based Detection of Influenza Compared to Prodesse ProFlu+ and Viral Culture.

Rapid and accurate diagnosis of influenza is important for patient management and infection control. We determined the performance of the cobas(®) Influenza A/B assay, a rapid automated nucleic acid assay performed on the cobas(®) Liat System for qualitative detection of influenza A and influenza B from nasopharyngeal (NP) swab specimens. Retrospective frozen and prospectively collected NP swabs from patients with signs and symptoms of influenza collected in universal transport medium (UTM) were tested at multiple sites including CLIA-waived sites using the cobas® Influenza A/B assay. Results were compared to the Prodesse Pro-Flu+ assay and to viral culture. Compared to the Prodesse ProFlu+ Assay, sensitivities of the cobas(®) Influenza A/B assay for influenza A and B were 97.7 and 98.6%, respectively; specificity was 99.2 and 99.4%. Compared to viral culture, the cobas(®) Influenza A/B assay showed sensitivities of 97.5 and 96.9% for influenza virus A and B, respectively; specificities were 97.9% for both viruses. Polymerase chain reaction (PCR)/sequencing showed that the majority of viral culture negative but cobas(®) Influenza A/B positive results were true positive results, indicating that the cobas(®) Influenza A/B assay has higher sensitivity compared to viral culture. In conclusion, the excellent accuracy, rapid time to result, and remarkable ease of use make the cobas(®) Influenza A/B nucleic acid assay for use on the cobas(®) Liat System a highly suitable point-of-care solution for the management of patients with suspected influenza A and B infection.

Stillbirth history and Toxoplasma gondii infection in women attending public health centers in a northern Mexican City.

Through a cross-sectional study design, 150 women attending public health centers with a history of stillbirths were examined for anti-Toxoplasma gondii IgG and IgM antibodies in Durango City, Mexico. Bivariate and multivariate analyses were used to assess the association of T. gondii seropositivity with the characteristics of the women with stillbirth history. Of the 150 women (mean age: 32.09 ± 9.16 years) studied, 14 (9.3%) had anti-T. gondii IgG antibodies and six (42.9%) of them were also positive for anti-T. gondii IgM antibodies. Multivariate analysis showed that T. gondii seropositivity was associated with high frequency (4-7 days a week) of eating meat (OR = 5.52; 95% CI: 1.48-20.59; P = 0.01), history of lymphadenopathy (OR = 4.52; 95% CI: 1.14-17.82; P = 0.03), and history of surgery (OR = 8.68; 95% CI: 1.04-72.15; P = 0.04). This is the first study on the seroepidemiology of T. gondii infection in women with a history of stillbirths in Mexico. The association of T. gondii exposure with a history of surgery warrants for further research. Risk factors for T. gondii infection found in the present survey may help to design optimal educational programs to avoid T. gondii infection.

Immune responses to Toxoplasma gondii in the gut.

Toxoplasma gondii is an important cause of disease, in pregnant women, newborns and immunocompromised hosts. Infection with T. gondii naturally occurs through ingestion of raw or undercooked meat containing cysts or through contact with cat feces containing oocysts. The intestine thus appears to be the site of invasion of the parasite. Since acute infection in immunocompetent hosts usually goes unnoticed, no information is available regarding the involvement of the intestine during acute infection in humans. However, in murine models of the disease the acute phase of infection has not been investigated in detail as well. In this report, an overview of the current knowledge regarding the initial phase of infection with T. gondii is given. Results of studies focussing on invasion and dissemination of the parasite and the immune responses elicited in the intestine are reviewed. The impact on the induction of local immune responses of the genetic background and sex of mice as well as the role of different doses and strains of the parasite are discussed. Finally, reports on the development of intestinal pathology in different animal species and the involvement of the intestine in human disease are reviewed.

A case of disseminated toxoplasmosis-value of PCR for the diagnosis.

Extracerebral toxoplasmosis has recently gained greater attention as a consequence of the AIDS epidemic. Serological techniques are unreliable, while isolation of the parasite is either time-consuming or insensitive. We here report a case of disseminated toxoplasmosis in a patient with AIDS. Diagnosis was suggested by serological tests and confirmed by PCR and Southern blot hybridisation or nested PCR. Detection of specific DNA was feasible in bronchoalveolar fluid, blood, serum and tissue samples. Direct detection of parasite-specific DNA by PCR and by nested PCR proved to be a promising, sensitive and rapid method for the diagnosis of disseminated toxoplasmosis, enabling us to promptly initiate specific treatment.

Effect of lipid matrix and size of solid lipid nanoparticles (SLN) on the viability and cytokine production of macrophages.

Solid lipid nanoparticles (SLN) interact with mononuclear cells following intravenous injection. Little is known about the interaction of SLN with these cells, including cytotoxic effects and a possible up-regulation of pro-inflammatory cytokines. Therefore, we investigated the influence of lipid matrix, concentration, and size of SLN on murine peritoneal macrophages (mphi). mphi were incubated with SLN consisting of different lipid matrices and coated with the same surfactant. Cytotoxicity as assessed by MTT test was found to be concentration-dependent and was dramatically influenced by the lipid matrix. Marked cytotoxic effects were observed when cells were incubated with SLN consisting of stearic acid (STE) or dimethyl-dioctadecylammonium bromide (DDA) at concentrations of 0.01%, whereas SLN consisting of triglycerides, cetylpalmitate or paraffin did not exert major cytotoxic effects at the same concentrations. Cytotoxic effects were most likely caused by products of enzymatic degradation including free stearic acid. Analysis of cytokine production by mphi following incubation with SLN revealed concentration-dependent decreases in IL-6 production. These decreases seemed to be associated with cytotoxic effects. IL-12 and TNF-alpha production was neither detected in supernatants of mphi treated with SLN at any concentration nor in those of untreated cells. The size of SLN did neither affect cytotoxicity of SLN nor resulted in induction or digression of cytokine production by mphi. In conclusion, results of the present study revealed that the nature of the lipid matrix and the concentration of SLN dramatically impact cytotoxicity of SLN on mononuclear cells. Lipid matrices of SLN should therefore be carefully chosen and tested for later intravenous use.

Surfactant, but not the size of solid lipid nanoparticles (SLN) influences viability and cytokine production of macrophages.

After intravenous (i.v.) injection, solid lipid nanoparticles (SLN) interact with mononuclear cells. Murine peritoneal macrophages were incubated with SLN formulations consisting of Dynasan 114 coated with different surfactants. The present study was performed to examine the impact of surfactants, which are important surface defining components of SLN, on viability and cytokine production by macrophages. Cytotoxicity, as assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test, was strongly influenced by the surfactant used being marked with cetylpyridinium chloride- (CPC-) coated SLN at a concentration of 0.001% and further increased at SLN concentrations of 0.01 and 0.1%. All other SLN formulations -- containing Poloxamine 908 (P908), Poloxamer 407 (P407), Poloxamer 188 (P188), Solutol HS15 (HS15), Tween 80 (T80), Lipoid S75 (S75), sodium cholate (SC), or sodium dodecylsulfate (SDS) -- when used at the same concentrations reduced cell viability only slightly. None of the SLN formulations tested induced cytokine production but a concentration-dependent decrease of IL-6 production was observed, which appeared to be associated with cytotoxic effects. IL-12 and TNF-alpha were detected neither in supernatants of macrophages treated with SLN at any concentration nor in those of untreated cells. In contrast to the type of surfactant, the size of SLN was found neither to affect cytotoxicity of SLN nor to result in induction or digression of cytokine production by macrophages. In conclusion, testing the effects of surfactants on SLN on activity of macrophages is a prerequisite prior to in vivo use of SLN.