First case of cryptococcosis in a new species of bandicoot (Bandicota indica) caused by Cryptococcus neoformans var. grubii.

The first case of cryptococcosis caused by Cryptococcus neoformans var. grubii in a new species of bandicoot (Bandicota indica) is described. The animal was trapped in a bamboo thicket in a park located in the city of Jabalpur, India. On necropsy, pathological lesions were seen in the lungs and liver and C. neoformans var. grubii was isolated from the lungs, liver, kidneys, spleen and brain but not the heart or intestine. The soil of the animal's burrow and bamboo debris around it also revealed the presence of C. neoformans var. grubii. We hypothesize that the bandicoots may potentially act as sentinel animals for environmental human pathogenic Cryptococcus species.     

Role of natural killer cells in resistance to systemic cryptococcosis

These studies demonstrate that Cryptococcus neoformans infection induced a dose-dependent augmentation of splenic natural killer (NK) cell activity by bg/+, but not bg/bg mice. To directly assess the role of NK cells in resistance to C. neoformans, bg/+ and bg/bg mice were treated with anti-NK-1.1 monoclonal antibody (mAb). Anti-NK-1.1-treatment abrogated the augmented NK cell activity observed during C. neoformans infection in bg/+ mice. Anti-NK-1.1-treated bg/+ mice had higher C. neoformans colony forming units (CFU) in their lungs on days 3 and 7 after intravenous (i.v.) challenge than control bg/+ mice. Moreover, the number of C. neoformans CFU in the lungs of anti-NK-1.1-treated bg/+ mice on days 3 and 7 were similar to those observed for infected bg/bg mice. By day 14, however, no differences in C. neoformans CFU were evident in the lungs of anti-NK-1.1-treated and control bg/+ mice. Anti-NK-1.1-treatment did not alter either the growth of C. neoformans in the spleens, livers, kidneys, or brain of bg/+ mice or the susceptibility of bg/bg mice to systemic cryptococcosis. These studies suggest that NK cells do not play a role in resistance to systemic cryptococcosis in the spleen, but do appear to play an early, but transient role in resistance to C. neoformans in the lungs. Overall, congenital defects in polymorphonuclear neutrophils (PMNs) and macrophages (M phi s), in addition to defects in NK cells, contribute to the enhanced susceptibility of bg/bg mice to systemic cryptococcosis.     

T cell-mediated immunity in the lung: a Cryptococcus neoformans pulmonary infection model using SCID and athymic nude mice.

T cells are important in systemic anticryptococcal defenses, but a role in controlling an initial pulmonary infection has not been demonstrated. A murine model with intratracheal inoculation was developed to study the acquisition and expression of pulmonary T cell-mediated immunity against Cryptococcus neoformans. Infections with four strains of C. neoformans (305, 68A, 613D, and 52D) in two strains of mice (BALB/c and C57BL/6) were examined. Unencapsulated strain 305 and slowly growing strain 68A were readily controlled apparently by nonimmune pulmonary defenses, and no extrapulmonary dissemination was detected. Strain 613D grew progressively in the lungs and disseminated to the brain and spleen. Strain 52D initially grew rapidly in the lungs and disseminated to the spleen, but a clearance mechanism developed in the lungs after day 7 postinfection and in the spleen after day 28. SCID and athymic nude mice were unable to clear a strain 52D pulmonary infection, and a lethal disseminated infection occurred. Pulmonary clearance could be adoptively transferred into SCID mice infected with strain 52D by use of immune T cells from the spleen and lungs and hilar lymph nodes of infected immunocompetent donors. Furthermore, pulmonary clearance was almost 100-fold better in SCID mice that received immune T cells from the lungs and hilar lymph nodes than in those that received immune T cells from the spleen, even though equivalent levels of delayed-type hypersensitivity were transferred by both cell populations. These adoptive transfer studies suggested that the lung and hilar lymph node T cells from immune animals either are enriched in such a way as to mediate protective immunity or home to the lungs better than do splenic T cells.     

Administration of endotoxin associated with lipopolysaccharide tolerance protects mice against fungal infection

Lipopolysaccharide (LPS) pretreatment of mice resulted in a significantly enhanced survival after disseminated Cryptococcus neoformans infection. The survival was associated with reduced fungal burden in tissues. LPS-pretreated mice had lower levels of cytokines in blood, spleen, and lungs and higher levels in brain. Pentoxifylline abolished the beneficial effect of LPS pretreatment.     

Histopathologic and ultrastructural studies of disseminated cytomegalovirus infection in strain 2 guinea pigs

Inbred strain 2 guinea pigs developed severe disseminated disease during acute experimental guinea pig cytomegalovirus (GPCMV) infection. A high mortality rate (100%) resulted, with most animals dying between 10 and 14 days after high dose (7.5 X 10(5) TCID50) virus inoculation. Infectious virus was recovered from many tissues, including spleen, lungs, liver, pancreas, heart, adrenals, kidneys, and salivary glands. The rate of GPCMV isolation from these tissues ranged from 50 to 100%. Gross lesions were observed in the spleen, liver, and lungs. On histologic examination, lesions were also seen in many other organs, including heart, pancreas, kidneys, adrenals, brain, intestines, and salivary glands. Intranuclear viral inclusions were present in many cell types of various organs. Under electron microscopic examination, cells with viral inclusions were easily found in the spleen, and liver, but less readily in the lungs, kidneys, salivary glands, and other organs. Most of the intranuclear inclusions consisted of electron-dense fibrils (10 nm diameter), viral nucleocapsids (100 nm), and tubular structures (60 nm diameter). Dense bodies and enveloped dense virions containing single or multiple capsids were present in the cytoplasm of many infected cells. The morphologic developments of GPCMV in these visceral tissues of strain 2 guinea pigs resembled those seen in GPCMV-infected cultured guinea pig cells but differed from those observed in the infected salivary gland duct cells. Strain 2 guinea pigs are a useful animal model for studying disseminated infection in CMV-associated human diseases.     

Role of CD4+ T cells in a protective immune response against Cryptococcus neoformans in the central nervous system.

Cryptococcosis is a life-threatening disease caused by the encapsulated yeast, Cryptococcus neoformans. Although infection with C. neoformans is initiated in the lungs, morbidity and mortality is mostly associated with infections of the central nervous system (CNS). Individuals with deficiencies in cell-mediated immunity, such as patients with AIDS, are more susceptible to disseminated cryptococcosis, highlighting the importance of cell-mediated immunity and CD4+ T cells in host resistance against C. neoformans. Using a mouse model of cryptococcal meningoencephalitis, we have shown that immunization of mice with a cryptococcal antigen induced a protective immune response that crossed the blood-brain barrier and initiated an immune response directly in the CNS if C. neoformans was present. The regional protective response was characteristic of a Type-1 (Th1) response in the types of cells present at the site of infection and in the cytokines and chemokines expressed. Here, we extend those findings and report that CD4+ T cells are required for survival of immune mice infected directly in the brain with C. neoformans and sensitized CD4 + T cells can transfer partial protection to naive mice infected intracerebrally with C. neoformans. Furthermore, CD4 + T cells were also important for optimal infiltration of inflammatory cells at the site of infection and in the expression of cytokines and chemokines associated with protection in the brain. Lastly, CD4+ T cells were required for optimal regional production and secretion of IFNgamma and in the significantly increased expression of iNOS in C. neoformans-infected brains of immune mice.     

Complementation of a capsule deficient Cryptococcus neoformans with CAP64 restores virulence in a murine lung infection

Cryptococcosis is a systemic infection in humans caused by the opportunistic fungal pathogen, Cryptococcus neoformans. The infection usually presents as chronic meningoencephalitis, but infects via the respiratory tract. A polysaccharide capsule is a major virulence factor, which allows the yeast to resist host defenses. However, the essential role of the capsule in allowing it to resist host defenses during the initial lung infection has not been clearly shown. A mutant acapsular C. neoformans strain 602 was complemented with the CAP64 gene to obtain an encapsulated strain, TYCC38-602. TYCC38-602 persisted in the lungs of C.B-17 mice after intratracheal inoculation and disseminated to the brain, whereas the mutant acapsular 602 and the plasmid control transformant CIP3-602 strains grew less readily in the lung and were infrequently detected in the brain. T cell-mediated immunity, developed to the encapsulated organism, was required to control growth within the lungs and had a significant impact on numbers of yeasts detected in the brain. The parent acapsular strain, but not the transformant control, also required T cells for optimal inhibition of growth within the lung, but not for maintaining control of the colony-forming units (cfu) in the brain. In summary, the cryptococcal capsule plays an important role in lung virulence and dissemination to the brain, and intact immunity is required to control lung growth of the encapsulated yeast.     

Monoclonal antibodies to Cryptococcus neoformans capsular polysaccharide modify the course of intravenous infection in mice.

Immunoglobulin G1 (IgG1) monoclonal antibodies (MAbs) to the capsular glucuronoxylomannan (GXM) were studied for their ability to modify the course of intravenous Cryptococcus neoformans infection in mice. A/J mice were given intraperitoneal injection of 1.0 mg of either a GXM-binding IgG1 MAb (2H1 or 2D10 gamma 1) or the irrelevant isotype-matched control MAb 36-65 prior to intravenous infection. Parameters used to study antibody efficacy were lung and brain tissue fungal burden, lung and brain weights, serum GXM levels, and histopathological examination of lung, brain, heart, kidney, and spleen tissues. Mice given GXM-binding MAb had significantly reduced lung tissue fungal burden as measured by CFU. In contrast to the reduction in lung tissue burden, the reduction in brain tissue burden was small and did not achieve statistical significance. Serum GXM levels were reduced in mice receiving GXM-binding MAb. Histopathological examination revealed reduced numbers of granulomas and C. neoformans organisms in the lungs, brains, and kidneys of MAb 2H1-treated mice relative to control mice. The lungs and brains of mice receiving GXM-binding MAb weighed significantly less than those of control animals, consistent with the reduced inflammation noted histologically. Subendocardial inflammation and kidney cortical infarctions were present in control infected mice but not in MAb 2H1-treated mice. Immunocytochemical staining for polysaccharide antigen revealed a marked reduction in the amount of tissue polysaccharide in mice treated with MAb 2H1 relative to control mice. The results support an useful role for passive antibody administration in C. neoformans infections.     

Role of natural killer cells in resistance to Cryptococcus neoformans infections in mice.

Previous studies have suggested a possible role for natural killer (NK) cells in resistance to some fungal infections, including Cryptococcus neoformans infections. The role of NK cells in early clearance of C neoformans from tissues and in long-term survival was studied in mice following intravenous inoculations of the organism. Mice treated with anti-asialo GM1 antiserum to temporarily reduce NK activity demonstrated an increase in colony-forming units (CFU) of C neoformans in the lung 24 hours after an intravenous inoculation of the organism. CFU in liver, spleen, kidney, and brain were not different in anti-asialo GM1 antiserum-treated versus control mice. An NK-specific reagent, anti-NK 1.1 monoclonal antibody, was used to deplete mice of NK cells in vivo for at least 14 days without affecting other natural defenses. The number of C neoformans retained in the lungs 24 hours after inoculation of the organism was significantly greater in NK cell-depleted mice than in controls, although CFU in other organs were unaffected. Following the intravenous inoculation of C neoformans, the survival of anti-NK 1.1-treated mice was not different from control mice. The effect of NK cell activity on resistance to C neoformans was also determined after an intratracheal inoculation of the organism. Mice pretreated with anti-NK 1.1 demonstrated no increases in CFU in the lungs, spleen, or brain as compared with controls. These data indicate that NK cells can play a role in vivo in early resistance against C neoformans if the organism is delivered via the intravenous route. However, NK cells do not play a role in either determining survival after an intravenous inoculation nor in resistance during an infection acquired via the respiratory tract.     

Cell wall targeting of laccase of Cryptococcus neoformans during infection of mice

Laccase is a major virulence factor of the pathogenic fungus Cryptococcus neoformans, which afflicts both immunocompetent and immunocompromised individuals. In the present study, laccase was expressed in C. neoformans lac1Delta cells as a fusion protein with an N-terminal green fluorescent protein (GFP) using C. neoformans codon usage. The fusion protein was robustly localized to the cell wall at physiological pH, but it was mislocalized at low pH. Structural analysis of the laccase identified a C-terminal region unique to C. neoformans, and expression studies showed that the region was required for efficient transport to the cell wall both in vitro and during infection of mouse lungs. During infection of mice, adherence to alveolar macrophages was also associated with a partial mislocalization of GFP-laccase within cytosolic vesicles. In addition, recovery of cryptococcal cells from lungs of two strains of mice (CBA/J and Swiss Albino) later in infection was also associated with cytosolic mislocalization, but cells from the brain showed almost exclusive localization to cell walls, suggesting that there was more efficient cell wall targeting during infection of the brain. These data suggest that host cell antifungal defenses may reduce effective cell wall targeting of laccase during infection of the lung but not during infection of the brain, which may contribute to a more predominant role for the enzyme during infection of the brain.     

Cryptococcosis outbreak in psittacine birds in Brazil.

An outbreak of cryptococcosis occurred in a breeding aviary in S?o Paulo, Brazil. Seven psittacine birds (of species Charmosyna papou, Lorius lory, Trichoglossus goldiei, Psittacula krameri and Psittacus erithacus) died of disseminated cryptococcosis. Incoordination, progressive paralysis and difficulty in flying were seen in five birds, whereas superficial lesions coincident with respiratory alterations were seen in two birds. Encapsulated yeasts suggestive of Cryptococcus sp. were seen in faecal smears stained with India ink in two cases. Histological examination of the birds showed cryptococcal cells in various tissues, including the beak, choana, sinus, lungs, air sacs, heart, liver, spleen, kidneys, intestines and central nervous system. High titres of cryptococcal antigen were observed in the serum of an affected bird. In this case, titres increased during treatment and the bird eventually died. Yeasts were isolated from the nasal mass, faeces and liver of one bird. Cryptococcus neoformans var. gattii serovar B was identified based on biochemical, physiological and serological tests. These strains were resistant (minimum inhibitory concentration 64 microg/ml) to fluconazole. This is the first report of C. neoformans var. gattii occurring in psittacine birds in Brazil.     

Regulatory Effects of Macrophage Inflammatory Protein 1α/CCL3 on the Development of Immunity to Cryptococcus neoformans Depend on Expression of Early Inflammatory Cytokines

Macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3 prevents the development of eosinophilic pneumonia (EP) driven by a nonprotective T2-type immunity during infection with a highly virulent strain of Cryptococcus neoformans. The present study evaluated the interaction of MIP-1alpha with other innate immune system cytokines by comparing the immune responses that followed pulmonary infections with high- (C. neoformans 145A) and low (C. neoformans 52D)-virulence strains. In contrast to what was found for C. neoformans 145A infection, lack of MIP-1alpha in C. neoformans 52D infection did not cause the development of EP. C. neoformans 52D induced tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), and MCP-1 in the lungs of infected wild-type (WT) and MIP-1alpha knockout (KO) mice by day 7 postinfection. Both WT and MIP-1alpha KO mice subsequently cleared this infection. Thus, the robust expression of early inflammatory cytokines in C. neoformans 52D-infected mice promoted the development of protective immunity even in the absence of MIP-1alpha. Alternatively, C. neoformans 145A-infected WT and MIP-1alpha KO mice had diminished TNF-alpha, IFN-gamma, and macrophage chemoattractant protein 1 (MCP-1) responses, indicating that virulent C. neoformans 145A evaded early innate host defenses. However C. neoformans 145A-infected WT mice had an early induction of MIP-1alpha and subsequently did not develop EP. In contrast, C. neoformans 145A-infected MIP-1alpha KO mice developed EP and had increased C. neoformans dissemination into the brain by day 35. We conclude that, in the absence of other innate immune response effector molecules, MIP-1alpha is crucial to prevent the development of EP and to control C. neoformans dissemination to the brain.     

Apoptosis induction by glucuronoxylomannan of Cryptococcus neoformans.

We studied the ability of glucuronoxylomannan (GXM), the major constituent of Cryptococcus neoformans capsular polysaccharide, to induce apoptosis in lymphocytes from normal rats. Spleen mononuclear cells (Smc) from normal rats treated with GXM for 24 h exhibited, in comparison with controls, an increased hypodiploidy in the DNA profile after staining with propidium iodide, as well as increased ladder-type DNA fragmentation in agarose gel electrophoresis and a high number of positive cells in the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) reaction. Furthermore, increased hypodiploidy in the DNA profile was also observed in Smc expressing T-cell receptor (TCR +). We also studied the induction of apoptosis in lungs and spleens from rats in the immunosuppressor period of disseminated cryptococcosis. TUNEL labeling of lungs and spleens from rats obtained 14 days after infection with C. neoformans showed a large number of apoptotic cells. Our results provide strong cytometric, molecular and morphological evidence that apoptosis could be a previously unrecognized immunosuppressive property of GXM in vitro. This programmed cell death may be involved in the immunosuppression observed during C. neoformans infection.     

CpG oligodeoxynucleotides promote the host protective response against infection with Cryptococcus neoformans through induction of interferon-gamma production by CD4+ T cells

In the present study, we elucidated the effect of synthetic CpG-containing oligodeoxynucleotides (ODN) on pulmonary and disseminated infection caused by Cryptococcus neoformans. CDF-1 mice were inoculated intratracheally with a highly virulent strain of this pathogen, which resulted in massive bacterial growth in the lung, dissemination to the brain and death. Administration of CpG-ODN promoted the clearance of C. neoformans in the lungs, decreased their dissemination to brain and prolonged the survival of infected mice. These effects correlated well with the enhanced production of interleukin (IL)-12 and interferon (IFN)-gamma and attenuated secretion of IL-4 in bronchoalveolar lavage fluids (BALF) and promoted development of Th1 cells, as indicated by the increased production of IFN-gamma by paratracheal lymph node cells upon restimulation with cryptococcal antigens. The IFN-gamma synthesis in BALF was inhibited by depletion of CD8(+) and CD4(+) T cells on days 7 and 14 after infection, respectively, but not by depletion of NK and gammadelta T cells. Consistent with these data, intracellular expression of IFN-gamma was detected predominantly in CD8(+) and CD4(+) T cells in the lung on days 7 and 14, respectively. The protective effect of CpG-ODN, as shown by the prolonged survival, was completely and partially inhibited by depletion of CD4(+) or CD8(+) T cells, respectively, but not by depletion of other cells. Finally, TNF-alpha was markedly induced by CpG-ODN, and the protective effect of this agent was strongly inhibited by neutralizing anti-TNF-alpha MoAb. Our results indicate that CpG-ODN alters the Th1-Th2 cytokine balance and promotes host resistance against infection with C. neoformans.     

Histopathological aspects of neurocryptococcosis in HIV-infected patients: autopsy report of 45 patients

The authors describe the histopathological necropsy findings of 45 human immunodeficiency virus (HIV)-infected patients with neurocryptococcosis. Systemic cryptococcosis with involvement of multiple organs such as spleen, liver, and lungs was present in all patients. Predominant diffuse meningoencephalitis predominantly in the basal ganglia, thalamus, and mid-brain, with minimal inflammatory infiltrate was seen in 30 AIDS patients (70%). We frequently observed in those patients the presence of multiple gelatinous pseudocysts with abundant Cryptococcus neoformans in the Virchow-Robin spaces and adjacent brain caused by the dissemination of the meningeal infection along the perivascular spaces. Isolated meningeal and cerebral involvement with minimal inflammatory infiltrate with numerous fungal organisms, and occasionally with granulomatous reaction and necrosis, was seen in 15 patients (30%). In addition to involvement of multiple organs by C. neoformans, HIV-infected patients with clinical manifestations of neurocryptococcosis frequently present a widespread involvement of the brain.     

Protective efficacy of antigenic fractions in mouse models of cryptococcosis

Infections due to the encapsulated fungus Cryptococcus neoformans are a significant cause of morbidity and mortality in patients with impaired T-cell function, particularly those with AIDS. Presumably then, T-cell responses to cryptococcal antigens are critical for protection against this ubiquitous fungus. To test the protective efficacy of these antigens as vaccine candidates, secreted cryptococcal antigens were separated by concanavalin A affinity chromatography into adherent (mannoprotein [MP]) and nonadherent (flowthrough [FT]) fractions, and the fractions were tested in murine models of disseminated cryptococcosis. Compared with adjuvant alone, C57BL/6 mice that received two inoculations of MP and FT exhibited prolonged survival and reduced brain and kidney fungal loads following intravenous challenge with C. neoformans strain B3501. MP-immunized animals had increased brain levels of tumor necrosis factor alpha, gamma interferon, and interleukin-2. Histopathologic examination revealed that compared with organs from mice that received only adjuvant, MP-immunized mice were able to recruit a stronger cellular infiltrate in brain, kidney, and liver in response to cryptococcal infection. Conjugated O-linked glycans were necessary for optimal MP-mediated protection, because chemical O deglycosylation reduced the protective efficacy of MP immunization. FT and MP immunization protected B-cell-deficient, but not T-cell-deficient mice, suggesting that protection was T-cell mediated. CBA/J mice also benefited from immunization with FT and MP, although the benefits were more modest than those seen with C57BL/6 mice. Thus, both MP and FT fractions of C. neoformans contain components that protect mice from disseminated cryptococcosis, and this protection appears to be T-cell mediated.     

Fulminant disseminated Varicella Zoster virus infection without skin involvement.

BACKGROUND: Varicella Zoster virus (VZV) infection is potentially very serious in bone marrow transplant recipients, and may manifest as a disseminated visceral infection. This condition is generally accompanied by a vesicular rash. OBJECTIVES: We review here a case of fulminant fatal disseminated VZV infection, not accompanied by skin involvement, and the laboratory approaches currently available to diagnose this disease. STUDY DESIGN: Post mortem tissue samples were subjected to histopathological examination, and tested for herpesviruses by electron microscopy and PCR. RESULTS: Intranuclear inclusions were noted by histological examination in the lungs, liver, kidneys and bone marrow. Particles with a herpesvirus morphology were visualized in liver tissue. VZV DNA was detected in liver and bone marrow by PCR followed by sequencing of the amplicons. Viremia was documented by retrospective testing of the serum by PCR. CONCLUSIONS: A disseminated VZV infection which proved rapidly fatal was demonstrated in a case without skin manifestations. This rare presentation of VZV infection is potentially underdiagnosed. Testing for VZV viremia by PCR can at the very least suggest the diagnosis although whether plasma-associated viremia is truly pathognomonic of visceral disseminated infection remains to be established.     

CRYPTOCOCCAL INFECTION AND TH1-TH2 CYTOKINE BALANCE

Cytokines have been recognized as key factors in determining host resistance to infectious pathogens. In particular, Th1-Th2 cytokine balance in hosts is profoundly associated with the outcome of infection caused by intracellular microbes. In a murine model of pulmonary and disseminated infection with Cryptococcus neoformans, an opportunistic fungal pathogen that frequently leads to fatal meningoencephaltis in severly immunocompromised hosts, expression of cytokine mRNA in the lungs from infected animals revealed Th2-dominant profiles, while administration of IL-12, which rescued mice from fatal infection, converted such balance toward Th1-dominant states in a drastic fashion. Thus, commitment of Th phenotypes critically determines host sensitivity to cryptococcal infection. In this review, we described how Th1-Th2 cytokine balance influences host protective responses to C. neoformans, and we identify the host and pathogen factors that regulate such balance.     

Cryptococcal infection and Th1-Th2 cytokine balance

Cytokines have been recognized as key factors in determining host resistance to infectious pathogens. In particular, Th1-Th2 cytokine balance in hosts is profoundly associated with the outcome of infection caused by intracellular microbes. In a murine model of pulmonary and disseminated infection with Cryptococcus neoformans, an opportunistic fungal pathogen that frequently leads to fatal meningoencephalitis in severely immunocompromised hosts, expression of cytokine mRNA in the lungs from infected animals revealed Th2-dominant profiles, while administration of IL-12, which rescued mice from fatal infection, converted such balance toward Th1-dominant states in a drastic fashion. Thus, commitment of Th phenotypes critically determines host sensitivity to cryptococcal infection. In this review, we described how Th1-Th2 cytokine balance influences host protective responses to C. neoformans, and we identify the host and pathogen factors that regulate such balance.     

Disseminated Conidiobolus infection with endocarditis in a cocaine abuser

A crack cocaine abuser developed disseminated infection caused by a species of Conidiobolus not known to cause disease in vertebrates. The fungus gained entry via skin abrasions on the lower extremities, spread through the hematogenous route, and caused endocarditis. There was evidence of fungal infection in the lungs, heart, kidneys, skeletal muscles, and brain. An additional complication was extensive rhabdomyolysis, with a marked elevation of creatine kinase of up to 1.2 million U/L.