Fusion Harboring Mast Cells Can Explain Molecular Positivity in Flow Cytometric MRD Negative Core Binding Factor AML.

Molecular measurable residual disease (MRD) can persist in core binding factor acute myeloid leukemia (AML) in otherwise disease-free patients. Utilizing cell sorting followed by fluorescent in situ hybridization, we show that detection is due to mast cells.

Measuring response to therapy in AML: Difference from normal flow cytometry vs RQ-PCR.

Multiple technologies have been used to monitor response to therapy in acute myeloid leukemia (AML) to improve detection of leukemia over the standard of practice, morphologic counting of blasts. The two techniques most frequently used in a routine clinical setting, flow cytometry and RQ-PCR, differ in their targets, sensitivity, and ability to detect residual disease. Both flow cytometry and RQ-PCR detect the expression of abnormal gene products, at the protein level or RNA level, respectively. Flow cytometry can be applied to a broad range of AML cases while RQ-PCR is limited to specific genetic abnormalities identified in subsets of AML. This article compares the results when both techniques were used in a reference laboratory to monitor AML over the course of treatment, comparing quantitative and qualitative results.

CD74 is expressed in a subset of pediatric acute myeloid leukemia patients and is a promising target for therapy: a report from the Children's Oncology Group.

As curative therapies for pediatric AML remain elusive, identifying potential new treatment targets is vital. We assessed the cell surface expression of CD74, also known as the MHC-II invariant chain, by multidimensional flow cytometry in 973 patients enrolled in the Children's Oncology Group AAML1031 clinical trial. 38% of pediatric AML patients expressed CD74 at any level and a comparison to normal hematopoietic cells revealed a subset with increased expression relative to normal myeloid progenitor cells. Pediatric AML patients expressing high intensity CD74 typically had an immature immunophenotype and an increased frequency of lymphoid antigen expression. Increased CD74 expression was associated with older patients with lower WBC and peripheral blood blast counts, and was enriched for t(8;21), trisomy 8, and CEBPA mutations. Overall, high CD74 expression was associated with low-risk status, however 26% of patients were allocated to high-risk protocol status and 5-year event free survival was 53%, indicating that a significant number of high expressing patients had poor outcomes. In vitro pre-clinical studies indicate that anti-CD74 therapy demonstrates efficacy against AML cells but has little impact on normal CD34+ cells. Together, we demonstrate that CD74 is expressed on a subset of pediatric AMLs at increased levels compared to normal hematopoietic cells and is a promising target for therapy in expressing patients. Given that nearly half of patients expressing CD74 at high levels experience an adverse event within 5 years, and the availability of CD74 targeting drugs, this represents a promising line of therapy worthy of additional investigation.

Evaluation of the procoagulant properties of a newly developed platelet modified lysate product.

BACKGROUND: Platelet transfusion is associated with logistical problems with the national storage guidelines of platelets. This results in decreased function in vivo as a result of the platelet storage lesion, and complications such as allergic or hemolytic reactions and thrombosis. We evaluated a new, freshly prepared platelet modified lysate (PML) product designed to be more procoagulant than fresh and stored platelets. METHODS: Fresh platelets were concentrated, sonicated, and centrifuged to produce PML. Samples of both washed and unwashed PML were evaluated for particle size, concentration, and activity, and then tested for clot kinetics and thrombin generation. PML samples were also stored at various temperatures for durations up to 6 months and evaluated for clot kinetics and thrombin generation throughout. RESULTS: PML showed significantly higher concentration of platelet microparticles, increased procoagulant properties, and increased thrombin generation as compared to fresh and stored platelets. In addition, PML maintained its clot kinetics over a 6-month storage period with variable storage conditions. CONCLUSIONS: The newly proposed PML product is more procoagulant, stable, and has additional potential applications than currently available platelet products. Further studies will be performed to assess its functions in vivo and to assess thrombotic potential.

Concurrent Polycythemia of Undetermined Etiology and Smouldering Plasma Cell Myeloma.

The combination of polycythemia and plasma cell myeloma occurring concurrently is very rare and few cases have been reported in the literature. Further, the vast majority of these cases are cases of polycythemia vera and myeloma. Here, we present a case of polycythemia of undetermined etiology and myeloma. The patient is a 48-year-old Caucasian male who was originally diagnosed with polycythemia of undetermined etiology. Twelve years later, when a bone marrow biopsy was performed in an attempt to determine the etiology of the polycythemia, findings diagnostic of plasma cell myeloma were discovered. Subsequent serum studies were also consistent with a plasma cell neoplasm, while evaluation for end-organ damage was negative. A battery of genetic and biochemical tests ruled out various congenital polycythemias, leading to a final diagnosis of polycythemia of undetermined etiology and smouldering plasma cell myeloma. This case highlights that while being unusual, polycythemia and plasma cell myeloma can occur concurrently, and, in this report, we discuss both entities and potential mechanisms of the pathophysiology of the concurrent presentation.

Emerging utility of flow cytometry in the diagnosis of chronic myelomonocytic leukemia.

The diagnosis of many hematologic malignancies has shifted from being based almost exclusively on morphology and clinical data to include ancillary studies such as flow cytometry. This trend has yet to affect the diagnosis of chronic myelomonocytic leukemia (CMML) as flow cytometry, while being integral in the diagnosis of many hematologic malignancies, has no explicit role in the current WHO criteria for CMML. The absence of WHO-determined criteria for flow cytometry in the diagnosis of CMML is not due to a lack of research on the subject over the years. Herein, we review the literature concerning the use of flow cytometry in the diagnosis of CMML, focusing on recent studies showing that CMML can be differentiated from other hematologic malignancies and reactive monocytoses by the quantification of monocyte subsets by flow cytometry with high sensitivity and specificity. We also detail how this methodology could be used clinically, both as a diagnostic test and potentially as a screening test.

Clinical Utility of Classical and Nonclassical Monocyte Percentage in the Diagnosis of Chronic Myelomonocytic Leukemia.

OBJECTIVES: To determine if a clinically applicable flow cytometry methodology could identify chronic myelomonocytic leukemia (CMML) cases. METHODS: Monocyte subset screening (CD14/CD16 expression) was performed on 68 blood and 25 bone marrow specimens with a monocytosis and/or flagged as possible CMML. Fifty thousand total events were obtained per case. Cases were categorized as CMML, atypical chronic myeloid leukemia (aCML), or non-CMML + non-aCML by clinicopathologic diagnosis. RESULTS: The methodology differentiated blood and bone marrow CMML cases from non-CMML + non-aCML but not three aCML cases in the clinical setting. Furthermore, a decreased percentage of nonclassical monocytes (CD14dimCD16+) showed better sensitivity than the previously described approach that relied on increased percentage of classical monocytes (CD14brightCD16-). CONCLUSIONS: Quantification of monocyte subsets is useful in clinical practice as a diagnostic marker of CMML in blood and bone marrow specimens. The percentage of nonclassical monocytes should be included in analysis of monocyte subsets.

The control of reactive oxygen species production by SHP-1 in oligodendrocytes.

We have previously described reduced myelination and corresponding myelin basic protein (MBP) expression in the central nervous system of Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) deficient motheaten (me/me) mice compared with normal littermate controls. Deficiency in myelin and MBP expression in both brains and spinal cords of motheaten mice correlated with reduced MBP mRNA expression levels in vivo and in purified oligodendrocytes in vitro. Therefore, SHP-1 activity seems to be a critical regulator of oligodendrocyte gene expression and function. Consistent with this role, this study demonstrates that oligodendrocytes of motheaten mice and SHP-1-depleted N20.1 cells produce higher levels of reactive oxygen species (ROS) and exhibit corresponding markers of increased oxidative stress. In agreement with these findings, we demonstrate that increased production of ROS coincides with ROS-induced signaling pathways known to affect myelin gene expression in oligodendrocytes. Antioxidant treatment of SHP-1-deficient oligodendrocytes reversed the pathological changes in these cells, with increased myelin protein gene expression and decreased expression of nuclear factor (erythroid-2)-related factor 2 (Nrf2) responsive gene, heme oxygenase-1 (HO-1). Furthermore, we demonstrate that SHP-1 is expressed in human white matter oligodendrocytes, and there is a subset of multiple sclerosis subjects that demonstrate a deficiency of SHP-1 in normal-appearing white matter. These studies reveal critical pathways controlled by SHP-1 in oligodendrocytes that relate to susceptibility of SHP-1-deficient mice to both developmental defects in myelination and to inflammatory demyelinating diseases.

Interferon-beta treatment in multiple sclerosis attenuates inflammatory gene expression through inducible activity of the phosphatase SHP-1.

Interferon-beta is a current treatment for multiple sclerosis (MS). Interferon-beta is thought to exert its therapeutic effects on MS by down-modulating the immune response by multiple potential pathways. Here, we document that treatment of MS patients with interferon beta-1a (Rebif) results in a significant increase in the levels and function of the protein tyrosine phosphatase SHP-1 in PBMCs. SHP-1 is a crucial negative regulator of cytokine signaling, inflammatory gene expression, and CNS demyelination as evidenced in mice deficient in SHP-1. In order to examine the functional significance of SHP-1 induction in MS PBMCs, we analyzed the activity of proinflammatory signaling molecules STAT1, STAT6, and NF-kappaB, which are known SHP-1 targets. Interferon-beta treatment in vivo resulted in decreased NF-kappaB and STAT6 activation and increased STAT1 activation. Further analysis in vitro showed that cultured PBMCs of MS patients and normal subjects had a significant SHP-1 induction following interferon-beta treatment that correlated with decreased NF-kappaB and STAT6 activation. Most importantly, experimental depletion of SHP-1 in cultured PBMCs abolished the anti-inflammatory effects of interferon-beta treatment, indicating that SHP-1 is a predominant mediator of interferon-beta activity. In conclusion, interferon-beta treatment upregulates SHP-1 expression resulting in decreased transcription factor activation and inflammatory gene expression important in MS pathogenesis.

Macrophages of multiple sclerosis patients display deficient SHP-1 expression and enhanced inflammatory phenotype.

Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of proinflammatory cytokine signaling, TLR signaling, and inflammatory gene expression. Furthermore, mice genetically lacking SHP-1 (me/me) display a profound susceptibility to inflammatory CNS demyelination relative to wild-type mice. In particular, SHP-1 deficiency may act predominantly in inflammatory macrophages to increase CNS demyelination as SHP-1-deficient macrophages display coexpression of inflammatory effector molecules and increased demyelinating activity in me/me mice. Recently, we reported that PBMCs of multiple sclerosis (MS) patients have a deficiency in SHP-1 expression relative to normal control subjects indicating that SHP-1 deficiency may play a similar role in MS as to that seen in mice. Therefore, it became essential to examine the specific expression and function of SHP-1 in macrophages from MS patients. Herein, we document that macrophages of MS patients have deficient SHP-1 protein and mRNA expression relative to those of normal control subjects. To examine functional consequences of the lower SHP-1, the activation of STAT6, STAT1, and NF-kappaB was quantified and macrophages of MS patients showed increased activation of these transcription factors. In accordance with this observation, several STAT6-, STAT1-, and NF-kappaB-responsive genes that mediate inflammatory demyelination were increased in macrophages of MS patients following cytokine and TLR agonist stimulation. Supporting a direct role of SHP-1 deficiency in altered macrophage function, experimental depletion of SHP-1 in normal subject macrophages resulted in an increased STAT/NF-kappaB activation and increased inflammatory gene expression to levels seen in macrophages of MS patients. In conclusion, macrophages of MS patients display a deficiency of SHP-1 expression, heightened activation of STAT6, STAT1, and NF-kappaB and a corresponding inflammatory profile that may be important in controlling macrophage-mediated demyelination in MS.

Modulation of macrophage infiltration and inflammatory activity by the phosphatase SHP-1 in virus-induced demyelinating disease.

The protein tyrosine phosphatase SHP-1 is a crucial negative regulator of cytokine signaling and inflammatory gene expression, both in the immune system and in the central nervous system (CNS). Mice genetically lacking SHP-1 (me/me) display severe inflammatory demyelinating disease following inoculation with the Theiler's murine encephalomyelitis virus (TMEV) compared to infected wild-type mice. Therefore, it became essential to investigate the mechanisms of TMEV-induced inflammation in the CNS of SHP-1-deficient mice. Herein, we show that the expression of several genes relevant to inflammatory demyelination in the CNS of infected me/me mice is elevated compared to that in wild-type mice. Furthermore, SHP-1 deficiency led to an abundant and exclusive increase in the infiltration of high-level-CD45-expressing (CD45(hi)) CD11b(+) Ly-6C(hi) macrophages into the CNS of me/me mice, in concert with the development of paralysis. Histological analyses of spinal cords revealed the localization of these macrophages to extensive inflammatory demyelinating lesions in infected SHP-1-deficient mice. Sorted populations of CNS-infiltrating macrophages from infected me/me mice showed increased amounts of viral RNA and an enhanced inflammatory profile compared to wild-type macrophages. Importantly, the application of clodronate liposomes effectively depleted splenic and CNS-infiltrating macrophages and significantly delayed the onset of TMEV-induced paralysis. Furthermore, macrophage depletion resulted in lower viral loads and lower levels of inflammatory gene expression and demyelination in the spinal cords of me/me mice. Finally, me/me macrophages were more responsive than wild-type macrophages to chemoattractive stimuli secreted by me/me glial cells, indicating a mechanism for the increased numbers of infiltrating macrophages seen in the CNS of me/me mice. Taken together, these findings demonstrate that infiltrating macrophages in SHP-1-deficient mice play a crucial role in promoting viral replication by providing abundant viral targets and contribute to increased proinflammatory gene expression relevant to the effector mechanisms of macrophage-mediated demyelination.

Induction of IL-33 expression and activity in central nervous system glia.

IL-33 is a novel member of the IL-1 cytokine family and a potent inducer of type 2 immunity, as mast cells and Th2 CD4+ T cells respond to IL-33 with the induction of type 2 cytokines such as IL-13. IL-33 mRNA levels are extremely high in the CNS, and CNS glia possess both subunits of the IL-33R, yet whether IL-33 is produced by and affects CNS glia has not been studied. Here, we demonstrate that pathogen-associated molecular patterns (PAMPs) significantly increase IL-33 mRNA and protein expression in CNS glia. Interestingly, IL-33 was localized to the nucleus of astrocytes. Further, CNS glial and astrocyte-enriched cultures treated with a PAMP followed by an ATP pulse had significantly higher levels of supernatant IL-1beta and IL-33 than cultures receiving any single treatment (PAMP or ATP). Supernatants from PAMP + ATP-treated glia induced the secretion of IL-6, IL-13, and MCP-1 from the MC/9 mast cell line in a manner similar to exogenous recombinant IL-33. Further, IL-33 levels and activity were increased in the brains of mice infected with the neurotropic virus Theiler's murine encephalomyelitis virus. IL-33 also had direct effects on CNS glia, as IL-33 induced various innate immune effectors in CNS glia, and this induction was greatly amplified by IL-33-stimulated mast cells. In conclusion, these results implicate IL-33-producing astrocytes as a potentially critical regulator of innate immune responses in the CNS.

Promoter-specific induction of the phosphatase SHP-1 by viral infection and cytokines in CNS glia.

We have previously shown that the protein tyrosine phosphatase SHP-1 is highly expressed in CNS glia and is an important modulator of cytokine signaling. As such, mice genetically lacking SHP-1 display constitutive myelin abnormalities, severe virus-induced demyelinating disease, and defects in innate anti-viral responses in the CNS. In this study, we show the differential distribution of the SHP-1 promoter-specific transcripts and demonstrate that several cytokines significantly induce SHP-1 expression in CNS glia. Consistent with these cytokine effects, infection with a neurotropic virus both in vitro and in vivo up-regulates SHP-1 transcripts and protein in CNS cells. Using CNS glial cultures of gene knockout mice, we show that interferons-beta and interferons-gamma act through STAT-1 and interferon regulatory factor-1 to induce the SHP-1 promoter I transcripts. Conversely, interferons-beta and IL-6 act through STAT-3 to induce SHP-1 promoter II transcripts. This study demonstrates that interferons and other cytokines associated with virus infections in the CNS can significantly induce the expression of SHP-1 through STAT-1/3 activity and provides a better understanding of the molecular mechanisms regulating cytokine-induced expression important for multiple homeostatic functions of SHP-1 in the CNS.

SHP-1 deficiency and increased inflammatory gene expression in PBMCs of multiple sclerosis patients.

Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of cytokine signaling, inflammatory gene expression, and demyelination in central nervous system. The present study investigates a possible similar role for SHP-1 in the human disease multiple sclerosis (MS). The levels of SHP-1 protein and mRNA in PBMCs of MS patients were significantly lower compared to normal subjects. Moreover, promoter II transcripts, expressed from one of two known promoters, were selectively deficient in MS patients. To examine functional consequences of the lower SHP-1 in PBMCs of MS patients, we measured the intracellular levels of phosphorylated STAT6 (pSTAT6). As expected, MS patients had significantly higher levels of pSTAT6. Accordingly, siRNA to SHP-1 effectively increased the levels of pSTAT6 in PBMCs of controls to levels equal to MS patients. Additionally, transduction of PBMCs with a lentiviral vector expressing SHP-1 lowered pSTAT6 levels. Finally, multiple STAT6-responsive inflammatory genes were increased in PBMCs of MS patients relative to PBMCs of normal subjects. Thus, PBMCs of MS patients display a stable deficiency of SHP-1 expression, heightened STAT6 phosphorylation, and an enhanced state of activation relevant to the mechanisms of inflammatory demyelination.

Chronic foot shock induces hyperactive behaviors and accompanying pro- and anti-inflammatory responses in mice.

Behavioral and accompanying physiological and immunological changes were investigated at various times during chronic irregular mild foot shock (CMFS) in adult male BALB/c mice. CMFS induced a significant hyperlocomotor activity in a familiar environment as well as increased consumption of chocolate milk (a favored drink) throughout the 5-week stress period. Unlike other chronic stress models, CMFS did not induce depressive-like behaviors. Hyperactivity was associated with transient elevations of pro-inflammatory cytokines (TNFalpha and IL-1beta) and IL-2 and more sustained (IL-10) or later (arginase activity) elevations in anti-inflammatory mediators in the spleen (serum levels below levels of detection) suggesting a transition from a pro-inflammatory state to an anti-inflammatory state during CMFS. Similar increases in brain levels of IL-2 and arginase activity were also detected and may contribute to CMFS-induced hyperactivity as both of these mediators have been shown to induce hyperactivity. To our knowledge, this is the first time that increased arginase activity has been documented during a stress paradigm. Altogether, the data indicate that CMFS induces behavioral changes distinct from other chronic stress models. CMFS is associated with multiple dynamic immunological changes, suggesting involvement of multiple factors in chronic stress-induced behavioral changes.

Antibody induction of lupus-like neuropsychiatric manifestations.

Although systemic lupus erythematosus (SLE) is usually evaluated with regard to autoimmune reactivity toward the kidney, there are multiple psychiatric abnormalities associated with this autoimmune disease. Lupus-prone male NZM88 mice, derived from NZB/NZW F1 mice, develop early neuropsychiatric manifestations without any signs of nephritis. In addition to the usual repertoire of antibody specificities, including autoantibodies to dsDNA and renal antigens, mice of this inbred strain express autoantibodies to numerous brain antigens. Here, we show that autoantibodies to brain antigens, assessed by Western analysis, are as individually varied as are the diverse neuropsychiatric manifestations observed in SLE patients. Additionally, a monoclonal antibody derived from the spleen of an untreated NZM88 male when injected into healthy BALB/cByJ, but not C57BL/6J, mice induced behaviors similar to those of lupus-prone NZM88 mice. This monoclonal antibody, which is specific to dynamin-1, binds preferentially in BALB/cByJ cortex and induces substantial expression of cytokines mainly in the hypothalamus. Thus, an antibody to just one brain antigen can induce multiple behavioral changes, and multiple autoantibodies to different brain antigens exist in lupus-prone mice; however, susceptibility to the induction of neurobehavioral deficits is dependent on host genetics.

Inverse regulation of inducible nitric oxide synthase (iNOS) and arginase I by the protein tyrosine phosphatase SHP-1 in CNS glia.

We have previously shown that the SH2 domain-containing protein tyrosine phosphatase SHP-1 plays a critical role in controlling virus infection in CNS glia in vivo and in vitro. The present study addressed whether increased virus replication in SHP-1-deficient glia in vitro may be a result of altered expression of inducible nitric oxide synthase (iNOS/NOS2). First, we observed a profound reduction in iNOS protein expression and production of nitric oxide (NO) in response to the viral mimic double-stranded RNA (dsRNA), despite the induction of high levels of iNOS mRNA, in SHP-1-deficient motheaten mouse compared to wild type littermate mouse glia. Because both iNOS expression and NO production are suppressed by multiple pathways involving arginase I activity, it was important that we observed abnormally high constitutive expression of arginase I in cultured glia of SHP-1-deficient compared to wild type mice. Further, both constitutive and IL-4/IL-10-induced expression of arginase I correlated with elevated STAT6 nuclear binding activity, decreased NO production, and increased virus replication in motheaten compared to wild type astrocytes. These findings provide the first evidence of an inverse relationship between NO and arginase I activity regulated by SHP-1 in CNS glia that is relevant to modulation of innate anti-viral responses. Thus, we propose that SHP-1 is a critical regulator of innate immunity to virus infections in CNS cells.

Regulation of avoidant behaviors and pain by the anti-inflammatory tyrosine phosphatase SHP-1.

The protein tyrosine phosphatase SHP-1 is a critical regulator of cytokine signaling and inflammation. Mice homozygous for a null allele at the SHP-1 locus have a phenotype of severe inflammation and are hyper-responsive to the TLR4 ligand LPS. TLR4 stimulation in the CNS has been linked to both neuropathic pain and sickness behaviors. To determine if reduction in SHP-1 expression affects LPS-induced behaviors, responses of heterozygous SHP-1-deficient (me/+) and wild-type (+/+) mice to LPS were measured. Chronic (4-week) treatment with LPS induced avoidant behaviors indicative of fear/anxiety in me/+, but not +/+, mice. These behaviors were correlated with a LPS-induced type 2 cytokine, cytokine receptor, and immune effector arginase profile in the brains of me/+ mice not found in +/+ mice. Me/+ mice also had a constitutively greater level of TLR4 in the CNS than +/+ mice. Additionally, me/+ mice displayed constitutively increased thermal sensitivity compared to +/+ mice, measured by the tail-flick test. Moreover, me/+ glial cultures were more responsive to LPS than +/+ glia. Therefore, the reduced expression of SHP-1 in me/+ imparts haploinsufficiency with respect to the control of CNS TLR4 and pain signaling. Furthermore, type 2 cytokines become prevalent during chronic TLR4 hyperstimulation in the CNS and are associated positively with behaviors that are usually linked to type 1 pro-inflammatory cytokines. These findings question the notion that type 2 immunity is solely anti-inflammatory in the CNS and indicate that type 2 immunity induces/potentiates CNS inflammatory processes.

Susceptibility of lupus-prone NZM mouse strains to lead exacerbation of systemic lupus erythematosus symptoms.

It has been repeatedly shown that the heavy metal mercury can induce or exacerbate lupus like autoimmunity in susceptible strains of rats and mice. A hallmark of such autoimmune induction is the accompaniment of an immune shift, in which there is usually an initial skewing toward a Th2-like immune environment. Another heavy metal, lead (Pb), has also been found to induce a Th2 shift in mice. However, exposure of normal mouse strains to Pb does not appear to induce autoimmunity. In order to investigate whether mice genetically predisposed to murine systemic lupus erythematosus (SLE) are susceptible to a Pb-induced exacerbation of lupus, males and females of four New Zealand mixed (NZM) mouse strains, along with BALB/c and C57Bl/6 controls, were administered three 100-microliter intraperitoneal injections of either 1.31 mM lead or sodium acetate per week for 3 wk. The four NZM strains chosen, NZM391, NZM2328, NZM88, and NZM2758, have differential genetic penetrance for SLE with variances in certain manifestations of the disease, but all of these strains naturally develop glomerulonephritis and produce high titers of anti-nuclear autoantibodies. The mice were prebled for baseline values and were bled directly after the injection period (d 1) and monthly thereafter for 5 mo. Sera were assessed for anti-double-stranded DNA titers, urea nitrogen levels, and creatine kinase activity, as well as four total immunoglobulin (Ig) G2a and IgG1 levels. Mortality and morbidity of the mice were also recorded. All NZM strains showed an acute, non-gender-based, susceptibility to Pb at d 1, but the control strains were unaffected. Over time, it became apparent that the strains diverged: The NZM391 strain showed gender-independent susceptibility to Pb enhancement of lupus manifestations and mortality; the NZM2328 strain exhibited gender-independent Pb susceptibility to manifestations, although only females had increased mortality; the NZM2758 strain exhibited non-gender-based elevations in urea nitrogen and creatine kinase activity levels; and the NZM88 strain displayed male susceptibility to anti-DNA and life span. Surprisingly, Pb increased the longevity of NZM88 and NZM2758 females. These results indicate that Pb indeed can exacerbate SLE in lupus-prone mice; however, even among lupus-prone strains, genetic differences determine the degree of exacerbation. Using the known phenotype and genetic differences, one can identify and characterize possible traits and loci associated with Pb susceptibility.