Effect of immunosuppressive drug regimens on acute and chronic murine toxoplasmosis

 To evaluate the potential risk of dissemination or reactivation of toxoplasmosis following the administration of immunosuppressive therapy we examined the effect of corticoids, azathioprine, and cyclosporine given alone or in combination on the course of murine acute and chronic toxoplasmic infection. Swiss Webster mice were infected perorally with a high-level inoculum of cysts of the C strain of Toxoplasma gondii. The evolution of the kinetics of parasite loads in the blood, brain, and lungs of infected and immunosuppressed mice was then sequentially followed. In mice with orally acquired infections initiated 2 days after the beginning of drug treatment, immunosuppression led to the persistence of parasites, especially in the lungs, which was most marked in mice treated with azathioprine and/or cortisol acetate. Administration of immunosuppressive therapy in mice previously infected with T. gondii resulted in a brief resurgence of parasite loads when treatment was started early after infection. Finally, under our experimental conditions we found that the immunosuppressive drugs that were given altered the natural course of infection with a prolonged persistence of parasites in the lungs but did not significantly affect parasite loads in the brain or lead to disseminated infection with detectable parasitemia. Received: 20 December 1995 / Accepted: 24 May 1996     

Intestinal colonization of a human subject by vancomycin-resistant Enterococcus faecium

Objective: To study the ability of two strains of vancomycin-resistant Enterococcus faecium to colonize the human intestine.
Methods: A single human subject ingested separately two strains of vancomycin-resistant E. faecium isolated from a pig and a chicken. The feces were cultured on selective medium. Prior to ingestion no vancomycin-resistant cocci were present in the feces. Ingestion of 10⁴-10⁵ CFU resulted in either no colonization or isolation only after enrichment. Ingestion of 10⁷ CFU of one strain resulted in colonization for a period of nearly 3 weeks, with fecal counts at times in excess of 10⁶ CFU/g. Ingestion of similar numbers of the other strain and reingestion of the first strain resulted in excretion in the feces for much shorter periods. When the fecal count of the ingested strains was greater than 10⁴-10⁵ CFU/g, the strains were isolated from swabs taken from perianal skin.
Conclusions: Vancomycin-resistant E. faecium strains from pigs and poultry are able to colonize the human gut and the perianal skin.     

Different virulence levels of the species of Sporothrix in a murine model

A comparative study on the experimental pathogenicity of five species of Sporothrix of clinical interest, Sporothrix albicans , Sporothrix brasiliensis , Sporothrix globosa , Sporothrix mexicana , and Sporothrix schenckii sensu stricto, was performed using an immunocompetent murine model. Two strains of each species and two levels of inoculum for each strain (2 × 10⁷ and 2 × 10⁴ conidia/animal) were tested by intravenous inoculation of mice (ten per group). Mortality was caused by the low inoculum of one strain of S. brasiliensis only, and the high inocula of S. brasiliensis and S. schenckii strains. Other inocula and other species tested did not kill any of the experimental animals. Tissue burden studies showed fungal spread to kidneys, lungs, spleen, brain, and testicles. S. brasiliensis was recovered extensively from all of the studied organs, and S. schenckii and S. globosa were recovered in lower amounts. Histopathological studies revealed differences in the lesions, which ranged from local inflammation with a low number of fungal cells at the injection site in mice infected with S. globosa , to massive infiltration of fungal cells in organs of those infected with S. brasiliensis . Our findings showed that S. brasiliensis and S. schenckii were the most virulent species, and suggest that lesional mechanisms could be species-specific.     

Immune privilege in sites of chronic infection: Leishmania and regulatory T cells

Summary:  Leishmania are digenetic protozoan parasites that are inoculated into the skin by vector sand flies, are taken up by macrophages, and produce a spectrum of chronic diseases in their natural reservoir and susceptible human hosts. During the early establishment of infection in the skin and lymphoid organs, Leishmania produce multiple effects on macrophage and dendritic cell functions that inhibit their innate anti-microbial defenses and impair their capacity to initiate T-helper 1 cell immunity. In addition, the skin is a site preconditioned for early parasite survival by virtue of a high frequency of steady-state, natural CD25⁺Foxp3⁺ regulatory T cells (Tregs) that function to suppress the generation of unneeded immune responses to infectious and non-infectious antigens to which the skin is regularly exposed. In murine models of infection, antigen-induced CD25^+/−Foxp3⁻interleukin (IL)-10⁺ Treg cells act during the effector phase of the immune response to control immunopathology and may also delay or prevent healing. Finally, following resolution of infection in healed mice, CD25⁺Foxp3⁺ Tregs function in an IL-10-dependent manner to prevent sterile cure and establish a long-term state of functional immune privilege in the skin.     

Impact of iron loading and iron chelation on murine tuberculosis

Objective: To demonstrate in a mouse model of tuberculosis that excess iron load enhances the growth of Mycobacterium tuberculosis and to assess whether or not iron chelation may abrogate the effect of iron loading on Mycobacterium tuberculosis growth in the mouse model.
Methods: In the first experiment, female BALB/C mice were infected intravenously with 5.4 × 10⁴ CPU of M. tuberculosis H37Rv per mouse. Before infection, half of them were treated for 2 weeks with 50 mg/kg polymaltose ferric hydroxide, a source of iron. In a second experiment, female BALB/C mice were infected intravenously with 9 × 10⁴ CPU per mouse and half of them were iron loaded for 2 weeks before infection. Both iron-loaded and non-iron-loaded mice were treated with desferrioxamine (DFO), an iron chelator, or isoniazid. At each sacrifice, mice and their spleens were weighed, lung lesions were noted, and the number of M. tuberculosis CFU determined by quantitative cultures of spleen and lungs.
Results: In the first experiment, the number of CFU was significantly higher in the spleen of iron-treated mice than in non-iron-loaded mice at days 14 and 28 after infection. In the second experiment, iron loading enhanced the multiplication of M. tuberculosis in the spleen but not in the lungs, DFO displayed a modest but significant effect on the multiplication of M. tuberculosis in iron-loaded mice, and isoniazid therapy was effective in both iron-loaded and non-iron-loaded mice.
Conclusions: Iron loading of BALB/C mice enhanced the multiplication of M. tuberculosis in the spleens but not in the lungs. DFO exhibited significant activity against M. tuberculosis in iron-loaded mice, and isoniazid therapy was strongly bactericidal in both iron-loaded and non-iron-loaded mice.     

Family and population studies of SAHH and ADA polymorphisms. A possible pitfall in the ascertainment of SAHH electrophoretic phenotypes

Typing of both SAHH and ADA red cell electrophoretic patterns was carried out among the members of about 80 families from Latium (Central Italy) and in a random sample of about 350 individuals from two Italian regions, Latium and Sardinia.
1. The SAHH ¹ enzyme product provided another interesting example of a change in the electrophoretic pattern brought about by the haemolysate ageing. In vitro storage of SAHH 1 red cell lysates leads to the production of a pattern similar to that expected from a heterozygote SAHH 2–1. This change has been shown to be abolished by pretreating the sample with mercaptoethanol.
The results indicate that the systematic use of sulphydril reducing agents can provide a more reliable means of analysing the SAHH polymorphism if differently stored samples are to be compared by starch gel electrophoresis.
2. Evidence against complete linkage of the SAHH and ADA loci has been obtained from two informative SAHH/ADA matings encountered in this study.
3. The SAHH allele frequencies observed in the two samples analysed were: SAHH ¹= 0.969, SAHH ²= 0.024, SAHH ³= 0.007 (Latium) and SAHH ¹= 0.973, SAHH ²= 0.011, SAHH ³= 0.016 (Sardinia).
4. The ADA ² allele frequency estimates were: 0.083 (Latium) and 0.059 (Sardinia). These figures are almost identical to those already reported for the same two regions.     

Population Dynamics of CD4+ T Cells Lacking Thy-1 in Murine Retrovirus-Induced Immunodeficiency Syndrome (MAIDS)

Increased numbers of CD4⁺ Thy-1 cells have been described in the spleen (SP) of mice with retrovirusinduced immunodeliciency (MAIDS). Since this phenotypic abnormality might have considerable functional importance, the expansion of the CD4⁺ Thy-1 subset in MAIDS was characterized further. CD4⁺ Thy-1⁻ and Thy-1⁺ T-cell is from infected mice expressed similar densities of CD3 and TCR γ/β. In contrast, the Thy-I⁻ subset was uniformly CD44^hi, even early in the disease when part of Thy-I⁺ cells were still CD44¹⁰. The emergence of CD4⁺ Thy-1⁻cells occurred first in SP and lymph nodes and was observed later in thymus. The important fraction ofCD4⁺ cells lacking Thy-1 normally present in Peyer's patches was only weakly modified. Despite the major expansion of the CD4⁺ Thy-1⁻ phenotype. the proliferating fraction was not higher in this subset than in CD4⁺ Thy-1⁺ cells from infeeted miee. Persistence after hydroxyurea administration was identical in both subsets, indicating similar mean cell lifespans. Taken together, these results show that the major expansion of CD4⁺ Thy-I⁻ T-cells in MAIDS is not ascribable solely to increased proliferation within this subset. Phenotypic analysis suggests that CD4⁺ Thy-I⁻ cells result from the differentiation of Thy-I⁺ cells induced by activation signals related to retroviral infection.     

Cytokine Dependence of Human to Mouse Graft-versus-Host Disease

Human peripheral blood leucocytes (PBL) induce chronic graft versus-host disease (GvHD) in non-conditioned severe combined immunodeficient mice. Chronic GvHD was observed in such animals after transplantation of 6 × 10⁷ human PBL per g body weight. However, acute xenogeneic GvHD results from grafting at least 2 × 10⁷ human PBL per g body weight to heavily conditioned murine hosts. The large numbers of human PBL were thought to be required fo produce above threshold amounts of certain cytokines. We show that treatment of the recipient mice with human interleukin 2 reduces the number of cells to inflict acute GvHD by a factor of ten. Human T cells and not B cells or macrophages, were previously shown to generate acute xenogeneic GvHD, when selected cell types from peripheral blood were grafted. Most of the infiltrating cells had the CD4⁺ phenotype. We demonstrate that CD4⁺ T cells are the main mediator, as the disease is abrogated by treating the mice with cytotoxic CD4 antibodies, but not with CD8 antibodies. A survival pattern, similar to that seen in GvHD, was induced by transplantation of a Herpesvirus saimiri transformed human CD4⁺ clonal T cell line in conjunction with daily interleukin 2 injections. Herpesvirus saimiri transformed human T cells allow easily reproducible graft properties in chimeric mouse models for human diseases.     

Pharmacokinetics of 18F-labeled trovafloxacin in normal and Escherichia coli-infected rats and rabbits studied with positron emission tomography

Objective: To measure tissue pharmacokinetics of trovafloxacin (CP 99,219) in normal and infected animals by both direct tissue radioactivity measurements and positron emission tomography (PET).
Method: Concentrations of [ ¹⁸ ]Ftrovafloxacin were measured in normal and infected rats ( n =6/group), at 10, 30, 60, and 120 min after injection, by radioactivity measurements. In normal rabbits ( n =4) and rabbits with Escherichia coli thigh infection ( n =4), tissue concentrations of drug were measured over 2 h with PET. After acquiring the final images, the rabbits were killed and tissue concentrations measured with PET were compared to the results of direct tissue radioactivity measurements.
Results: In both species, there was rapid distribution of [ ¹⁸ ]F trovafloxacin in most peripheral organs. Peak concentrations of more than five times the MIC₉₀ of most Enterobacteriaceae and anaerobes (>100-fold for most organisms) were achieved in all tissues and remained above this level for >2 h. Particularly high peak concentrations were achieved in the kidney (>75 μg/g), liver (>100 μg/g), blood (>40 μg/g), and lung (>10 μg/g). Even though the concentration of trovafloxacin in infected muscle was reduced ( p <0.01), the peak concentration was still >4 μg/g and tissue levels remained above 2 μg/g for more than 2 h. Due to the lower concentrations that were achieved in the brain (peak ˜5 μg/g), it is expected that trovafloxacin will have limited central nervous system toxicity.
Conclusions: PET with [¹⁸F] trovafloxacin is a useful technique for non-invasive measurements of tissue pharmacokinetics.     

Immunological Response of Major Histocompatibility Complex Class II-Deficient (Aβ°) Mice Infected by the Parasite Schistosoma mansoni

We have characterized the immunological behaviour of major histocompitibility complex (MHC) Class II molecule-deficient (Aβ°) mice after infection by Schistosoma mansoni . In Aβ° mice, morbidity developed dramatically 7 weeks after infection leading to death, despite the absence of an increase in parasite burden or of eggs trapped in the liver. Histological examination of the liver revealed the absence of a classical granulomatous reaction. Antibodies were produced only against schistosomulum antigens. Specific antibodies against adult worm (SWAP) or egg antigen (SEA) were not detected. Cytokine production (IFN-γ and IL-4) was absent after in vitro restimulation of splenic cells from infected Aβ° mice with parasite antigens. Adoptive transfer of primed splenic cells (total, purified CD4⁺ or CD8⁺ T cells) failed to improve survival or to induce a granulomatous reaction in infected Aβ° mice. Survival, cellular and humoral responses in CD8⁺ T-cell-depleted Aβ° mice or MHC° mice (lacking MHC class I and II molecules) were similar to nondepleted Aβ° mice, suggesting that anti-schistosomula antibody production was thymo-independent. Our results demonstrate a high degree of susceptibility of Aβ° mice to infection and corroborate the importance of CD4⁺ T cells in the initiation of the granulomatous response. However, our results do not show evidence for the involvement of CD8⁺ T cells in response to S. mansoni infection.     

Cytokine and chemokine expression in the skin from patients with maculopapular exanthema to drugs

Background:  Maculopapular exanthema (MPE) is the most frequent clinical manifestation of nonimmediate allergic reactions to drugs and T helper 1 (Th1) cytokines and CD4⁺ T cells have been shown to play an important role in its pathogenesis. We assessed the role of cytokines and chemokines and their receptors in the pathogenesis of MPE.
Methods:  We evaluated skin biopsies and peripheral CD4⁺ and CD8⁺ T cells from 27 patients during the acute phase of the reaction and 26 exposed controls. Semiquantitative real-time PCR was performed to determine the expression of cytokines and chemokines and their receptors and immunohistochemistry was used to determine the same chemokines and their receptor proteins in skin.
Results:  There was a high expression of the Th1 cytokines interferon-γ ( P  = 0.006) and tumor necrosis factor-α ( P  = 0.022) in skin and CD4⁺ T cells ( P  = 0.007 and P  = 0.005, respectively); and of the Th1 chemokines CXCL9 ( P  = 0.005) and CXCL10 ( P  = 0.028) in the skin, while their receptor CXCR3 was increased in skin ( P  = 0.006) and CD4⁺ T cells ( P  = 0.03). Homing chemokine receptors were also increased: CCR6 in skin ( P  = 0.026) and CD4⁺ T cells ( P  = 0.016), and CCR10 only in CD4⁺ T cells ( P  = 0.016), as well as their ligands, CCL20 and CCL27, in skin alone. Immunohistochemistry confirmed these results.
Conclusions:  These data show significant differences in the expression of chemokines and chemokine receptors, related with a Th1 profile, in both skin biopsies and peripheral CD4⁺ T cells in patients with drug-induced MPE.     

Lymphocytic Choriomeningitis Virus Infection is Associated with Long-Standing Perturbation of LFA-1 Expression on CD8+ T Cells

Flow cytometric analysis of splenocytes from mice infected with lymphocytic Choriomeningitis virus revealed marked and long-standing up-regulation of LFA-1 expression on CD8⁺, but not on CD4⁺ T cells. Appearance of CD8⁺ T cells with a changed expression of adhesion molecules reflected polyclonal activation and expansion which was demonstrated not to depend on CD4⁺ T cells or their products. Cell sorting experiments defined virus-specific CTL to be included in this population (LFA-1^hiMEL-14^lo), but since about 80% of splenic CD8⁺ T cells have a changed phenotype, extensive bystander activation must take place; this is indicated also by the finding that CD8⁺LFA-l^hi cells transiently express several markers of cellular activation, e. g. transferrin receptor, IL-2Rα and β. Analysis of cells from the cerebrospinal fluid of mice infected intracerebrally showed that virtually all T cells present belonged to the CD8LFA-lhi subset and, correspondingly, the ligand ICAM-1 was found to be up-regulated on endothelial cells in the inflamed meninges. Preincubation of LCMV-primed donor splenocytes with anti-LFA-1 markedly inhibited the transfer of virus-specific delayed-type hypersensitivity to naive recipients. Together, these findings indicate that up-regulation of LFA-1 expression is a critical factor involved in directing activated CD8⁺ T cells to sites of viral infection.     

Phenotypic Characterization of Macrophages in the Endometrium of the Pregnant Cow

Problem  Macrophages are recruited in large number to the interplacentomal endometrium of the cow during pregnancy. We evaluated whether endometrial macrophages also accumulate in placentomal regions of endometrium during pregnancy and whether endometrial macrophages are regionally differentiated.
Method of study  Interplacentomal endometrium and placentomes were subjected to dual-color immunofluorescence using CD68 as a pan-macrophage marker.
Results  CD68⁺ cells were abundant in stroma of the interplacentomal endometrium and caruncular septa of the placentomes. CD68⁺ cells were not present in fetal villi of the placentomes or in the interplacentomal chorion. Regardless of location, the majority of CD68⁺ cells also expressed CD14. In interplacentomal endometrium, CD68⁺CD11b⁺ cells were present in deeper areas of the stroma but not in shallow endometrial stroma. In caruncular septa of the placentome, CD68⁺ cells were negative for CD11b. CD68⁺ cells in the interplacentomal endometrium were negative for MHC class II while most CD68⁺ cells in caruncular septa were positive for MHC class II.
Conclusion  CD68⁺CD14⁺ macrophages present in the stroma of the interplacentomal endometrium and caruncular septa of the placentome are regionally differentiated with regard to expression of CD11b and MHC class II.     

Effects of Peritoneal Fluid from Endometriosis Patients on Interferon-γ-Induced Protein-10 (CXCL10) and Interleukin-8 (CXCL8) Released by Neutrophils and CD4+ T Cells

Problem  Intraperitoneal immuno-inflammatory changes may be associated with the pathogenesis of endometriosis. We evaluated the effects of peritoneal fluid obtained from patients with endometriosis (ePF) on the release of interferon-γ-induced protein-10 (IP-10/CXCL10) and interleukin-8 (IL-8/CXCL8) by neutrophils, CD4⁺ T cells, and monocytes.
Method of study  Neutrophils, CD4⁺ T cells, and monocytes were cultured with ePF and the chemokine levels in the supernatants were then measured using enzyme-linked immunosorbent assay.
Results  The addition of ePF to cultures of CD4⁺ T cells led to a significant increase in the release of IP-10 when compared with control PF without endometriosis (cPF). There was a positive correlation between the levels of IL-8 and IP-10 in ePF ( R  = 0.89, P  =   0.041), but not between the levels of IP-10 and IL-8 released by neutrophils, CD4⁺ T cells, and monocytes. The levels of IP-10 in ePF were positively correlated with the release of IP-10 by ePF-treated neutrophils ( R  = 0.89, P  <   0.001), CD4⁺ T cells ( R  = 0.93, P  <   0.001), and monocytes ( R  = 0.70, P  =   0.01). Moreover, the addition of ePF significantly enhanced the interferon-γ-induced release of IP-10 by nuetrophils and CD4⁺ T cells.
Conclusion  These findings suggest that neutrophils and T cells release differential levels of IP-10 and IL-8 in response to stimulation with ePF, and that these cells are a major source of IP-10 in the PF of endometriosis patients.     

MIC distribution and inoculum effect of LY333328: A study of vancomycin-susceptible and VanA-type and VanC-type enterococci obtained from intensive care unit patient surveillance cultures

Objectives: To determine the in vitro activity and inoculum effect of LY333328, a semisynthetic glycopeptide, against vancomycin-susceptible and vancomycin-resistant enterococcal isolates.
Methods: One hundred and seventy-six enterococcal isolates (117 vancomycin-susceptible, 29 VanA-type and 30 VanC-type isolates) obtained from surveillance cultures of 139 intensive care unit patients were studied by the standard agar dilution method. Vancomycin resistance determinants were characterized by PCR.
Results: The activity of LY333328 was comparable (MIC range, 0.1–2 mg/L) to those of vancomycin (0.1–4 mg/L) and teicoplanin (0.06–1 mg/L) for vancomycin-susceptible isolates. LY333328 was more active (0.1–8 mg/L) than vancomycin (256 to >1024 mg/L) and teicoplanin (32–512 mg/L) against VanA-type isolates, and similar (0.2–1 mg/L) to teicoplanin (0.1–0.5 mg/L) against VanC-type isolates. The MIC distribution of LY333328 displayed a narrower range than that of vancomycin, with no clear distinction between susceptible and resistant populations. The increment in the inoculum size, from 10⁴ to 10⁶ CFU/spot, of susceptible isolates increased the MIC values of LY333328, vancomycin and teicoplanin by factors of 11.4, 1.6 and 3.8, respectively. The corresponding factors for LY333328 for VanA-type and VanC-type isolates were 3.5 and 6.4, respectively.
Conclusions: LY333328 displays an excellent in vitro activity against vancomycin-susceptible and -resistant enterococci. Nevertheless, the inoculum size used in susceptibility tests should be carefully controlled.     

Control of intestinal inflammation by regulatory T cells

Summary: Transfer of CD4⁺ T cells to immune-deficient mice in the absence of the CD25⁺ subset leads to the development of colitis, indicating that regulatory cells capable of controlling a bacteria-driven inflammatory response are present in normal mice. Cells with this function are present in the thymus as well as in the periphery of germ-free mice, suggesting they may be reactive with self-antigen. These cells resemble CD4⁺CD25⁺ cells that inhibit organ-specific autoimmunity, suggesting that a similar subset of regulatory T cells may control responses to self and foreign antigens. Development of colitis is dependent on accumulation of activated CD134L⁺ dendritic cells (DC) in the mesenteric lymph nodes, which is inhibited by CD4⁺CD25⁺ cells, indicating that regulatory T cells may control DC activation in vivo . Whilst inhibition of T-cell activation in vitro by CD4⁺CD25⁺ cells does not involve interleukin-10 and transforming growth factor-β, these cytokines are required for the suppression of colitis. It may be that control of responses that activate the innate immune system requires multiple mechanisms of immune suppression. Recently, we identified CD4⁺CD25⁺ cells with immune suppressive activity in the thymus and peripheral blood of humans, raising the possibility that dysfunction in this mechanism of immune regulation may be involved in the development of autoimmune and inflammatory diseases.     

Cellular and Cytokine Characterization of Vascular Inflammation in CBA/J Mice Chronically Infected with Trypanosoma cruzi

Continuing our characterization of the immunopathological events occurring during experimental murine Chagas' disease, an immunohistological examination was conducted of the aortas of chronically infected CBA/J mice. Compared with non-infected mice of identical age, Trypanosoma cruzi -infected mice exhibited a marked vasculitis, with significant infiltration of inflammatory cells into the adventitial layer, including CD4⁺, CD8⁺ T cells and macrophages. Production of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) was evident in the inflammatory infiltrate in the endothelial and smooth muscle layers. Vasculitis was most apparent in proximity to the heart, but extended along the aorta. Such an inflammation could lead to an alteration of the endothelium, altering the protective properties of this layer and further contributing to the focal pathology characteristic of this stage of infection.     

Recognition of HLA-A1 by murine monoclonal antibodies

Abstract: Balb/c mice were immunized with cells from the mouse mastocytoma line P815 transfected with an HLA-A1 gene. The splenocytes of the immunized mice were fused with cells from the murine myeloma NS-1. In an initial screening, supernatants of growing cultures were tested for their binding capacity to the immunizing P815/A1⁺ cells as well as to P815/A2⁺ cells. Three out of 756 hybrids produced antibodies which bound to P815/A1⁺ cells only. They were cloned and further analyzed for their binding reactivity to reference B-lymphoblastoid cells from the Tenth International Histocompatibility Workshop. One monoclonal antibody, designated 6B11, reacted only with HLA-A1⁺ cells, while the two other antibodies, 3G3 and 7F10, appeared to detect antigenic determinants shared by HLA-A1, A3, A11, A26, and A30 (3G3) and by HLA-A1, A3, A11, A26, A28 and A30 (7F10). Flow cytometric studies on B-lymphoblastoid cell lines as well as on a series of tumor cell lines, including melanoma and colon carcinoma lines, confirmed the specificity of these antibodies. Monoclonal antibodies 7F10 and 6B11 were found to be of the IgM class and 3G3 of the IgG1 class. By complement-dependent ⁵¹Cr release experiments it was further shown that the two IgM antibodies 7F10 and 6B11 were able to lyse all cell lines of the HLA-A1 haplotype tested.