WO2009111476A2 - Assay for systematic lupus erthematosus - Google Patents

Abstract

An assay for the presence of systemic lupus erythematosus (SLE) is disclosed. In one aspect, a specific a specific bacterial nucleic acid sequence of one or more of SEQ ID NOs: 1-9 is admixed with an analyte from the human to be diagnosed and the presence or absence of nucleic acid hybrid formation is determined. In another embodiment, antibodies from the human to be diagnosed are admixed with a promyelocyte Ehrlichiae Ep group antigen to form an antigen/antibody admixture that is maintained for a time period sufficient for immunoreaction and formation of an immunoreaction product, and the presence or absence of the immunoreaction product is determined. In each respective instance, nucleic acid hybrid formation or immunoreaction product formation indicates that the human has SLE.

Description

ASSAY FOR SYSTEMATIC LUPUS ERTHEMATOSUS

TECHNICAL FIELD

This invention relates to the diagnosis of systemic lupus erythematosus in a human patient . More particularly, this invention relates to an assay for the presence of specific a specific bacterial nucleic acid sequence or antibodies in human tissues that indicates that a person has systemic lupus erythematosus (SLE) .

BACKGROUND ART

Systemic lupus erythematosus (SLE) is a severe disease characterized by chronic inflammation (swelling, redness, and pain) . Patients having this disease produce antibodies that target cells of their own body tissues. The antibody-targeted cells are then destroyed by white blood cells in the body causing cell death that leads to inflammation. As such, SLE is known as an autoimmune disease. Crow et al . , "Etiologic Hypothesis for Systemic Lupus Erythematosus, " in LaHita, Systemic Lupus Erythematosus , Churchill, Livingston, N. Y. (1987) page 51 ff . SLE affects many body systems including skin, joints, blood, lungs, kidneys, heart, brain, gastrointestinal tract, bone marrow, liver, and nervous tract. Crow et al . , "Etiologic Hypothesis for Systemic Lupus Erythematosus, " in LaHita, Systemic Lupus Erythematosus, Churchill, Livingston, N. Y. (1987) page 51 ff.

The present diagnosis for SLE is an art form practiced by rheumatologists or other physicians. In one form, the practitioner seeks eleven indicia: malar rash, discoid rash, oral ulcers, arthritis, serositis, renal disorder, neurologic disorder, hematologist disorder, immunologic disorder and anti-nuclear antibodies (ANA) . SLE is presumed if four of the eleven are present. LaHita, Systemic Lupus Erythematosus, 2nd. ed., Churchill, Livingston, N. Y. (1992) pages 372- 373. Other assays that can be and are used to diagnose the presence of SLE in a patient include separate assays for ANA, complement levels, the presence of rheumatoid factor, hyperglobulinemia, false positive syphilis test, LE cell test, anti-DNA antibodies, anti-SMP antibodies, anti-RMP antibodies, anti-Smith antibodies, anemia, leucopenia, thrombocytopenia, positive direct Coombs' test, and anti-double-stranded DNA (anti-dsDNA) antibodies. LaHita, Systemic Lupus Erythematosus, 2nd. ed. , Churchill, Livingston, N. Y. (1992) . As is seen, none of these assays is definitive for the disease.

Although there is marked similarity to an infectious entity, an exhaustive search for an etiologic agent has not until recently yielded any candidates that fulfill the criteria for causation of this disease until now. Crow et al., "Etiologic Hypothesis for Systemic Lupus Erythematosus," in Lahita, Systemic Lupus Erythematosus, Churchill, Livingston, N. Y. (1987) page 51 ff; Pincus, Arthr. & Rheum., 20:149-158 (1982) . More recently, the consideration of bacteria and mycoplasmas with unique capacities to perturb immune systems has led to new hypotheses in regard to the infectious trigger of SLE. For example, intra-erythrocyte organisms with characteristics that were thought to be Haemobartonella-like were first suggested as exogenous agents in SLE by Kallick et al . , Nature New Biology, 236:145-146 (1972) . That report was further developed by a later report of antigenic similarities between SLE or lupus nephritis and diseases caused by Anaplasma marginale, an intra-erythrocytic parasite of cattle, and a member of the family Anaplasmataceae. Kallick et al . , Arthr. Rheum., 23:197-205 (1980) .

Further, exogenous intra-erythrocytic structures seen in the same erythrocyte by Giemsa or acridine orange staining and phase contrast microscopy have been observed in patients with SLE, and are illustrated in U.S. Patent No. 5,972,309 and U.S. Patent No. 5,795,563. These stained structures are identical or similar in appearance to Mycoplasma haemofelis, the causative agent of feline infectious anemia, as discussed below.

A specific group of Mycoplasmas recognized as Haemoplasmas also produce hematological disease in cats, dogs, mice, and swine. They exhibit latency and chronicity as well as an acute syndrome and intermittent infection of the erythrocytes and can be seen in the acute phase of early infection, although not easily in the chronic phase. They have not been reproducibly cultured or transmitted to other than related animal species.

The locus of infection of the erythrocyte in the Haemoplasmas , M. haemocanis , M. haemofelis, and M. haemosuis is seen on the surface of the erythrocytes, though in mice, M. haemomuris also known as Haemobartonella muris, is within the erythrocyte. Early descriptions of these agents were mediated by the observed position of the parasite on the erythrocyte .

In animal disease, antibiotics control hemolytic anemia, the primary pathologic event, only if given early in the course of the illness. Late in the course of animal illness, the hemolytic anemia is mediated by antibody formation and antibiotics do not appear to affect the course. In veterinary literature, one antibiotic that appears to suppress some of the clinical manifestations is tetracycline and analogues. Franklin et al., Southwestern Vet., 15:131-139 (1962) .

Several humans with SLE or connective tissue disease have been treated with tetracycline (doxycycline) in preliminary work of the inventor based on the presumption of Anaplasmataceae parasitemia. Wanduragala et al. in, Rickettsial and Chlamydial Diseases of Domestic Animals, Waldehewit ed. , Pergamon Press, Oxford, (1993) page 79.

Aureomycin, a tetracycline- like drug, had been proposed as a treatment in the 1940s for rheumatoid diseases with claims of some degree of success. [Brown et al . , J. Lab. Clin. Med., 34:1404- 1410 (1949); Scheff et al . , Infec. Dis., 98:113 (1956) .] These phenomena suggest that the tetracycline drugs are of benefit in the syndrome of SLE.

Current SLE therapy relies upon heavy steroid use concurrent with immunosuppressives and/or plasmapheresis . It is of interest that infections in animals by bacteria then called Anaplasmataceae, are almost uniquely among infectious diseases, ameliorated by steroids. [Scheff et al . , Infec. Dis., 98:113 (1956); Ristic et al . , J. Vet. Res., 19:37 (1958)]

No presently used therapy is completely satisfactory. Although the life expectancy of lupus patients has been considerably increased, the ravages of therapeutic side effects and the constant fatigue take a severe toll. Dubois, Lupus Erythematosus , 2d ed., U.S. California Press, Los Angeles (1974) .

US application Serial No. 11/461900 that was published as application 2007003182 on February 08, 2007 by the present inventor teaches that lupus is also a disease of the marrow and blood progenitor cells. As such, lupus can be assayed for in megakaryocytes of the bone marrow where precursors to the various circulating bloods cells and such circulating cells can be found. The bone marrow of a person suspected of having SLE is assayed for the presence of specific bacterial structures within those cells. Those internal bacterial structures specifically stain with a dye such as a DNA intercalating dye like Giemsa, ethidium bromide, acridine orange, or the like as are well known in the art. The presence of those specifically stainable structures within the patient's megakaryocytes indicates that the patient has SLE. Ehrlichiae are obligate intracellular bacteria that predominantly infect bone marrow-derived cells in their mammalian hosts. Those species, for which a biological vector is known, are transmitted by ticks. Typically, Ehrlichiae are contained within a membrane-lined vacuole of their host cell. Ehrlichiae are a group of pathogens related to Amaplasma marginale. The use of E. phagocytophila in the diagonosis of human granulocytic ehrlichiosis is disclosed in US Patent No. 5,928,879.

Ehrlichiae species were initially characterized on the basis of host cell type, host species, and serologic cross-reactivity. Ehrlichiae can be divided into three phylogenetically distinct groups on the basis of nucleotide sequences of the 16S ribosomal RNA genes in each species and strong serologic cross-reactions. Each group is denoted by the historical precedent for the genetic group.

The Ehrlichia canis group includes 3 species known to infect predominantly monocytes and macrophages, and a single species known to infect canine granulocytes. Ehrlichia canis, the type species, E. chaffeensis , and E. muris infect mononuclear phagocytes of dogs, humans, and mice, respectively, whereas E. ewingii infects canine granulocytes [Ristic et al, In: Bergey's manual of Systematic Bacteriology, 1(9):1957 (1984)] .

The second genetic group, the E. sennetsu group, includes E. sennetsu and E. risticii . They are monocytic Ehrlichiae that are agents of human Sennetsu fever of Japan and Potomac horse fever (equine monocytic ehrlichiosis) of horses worldwide.

The third group, the E. phagocytophiIa (Ep) genogroup, includes the granulocytic Ehrlichiae, E. equi, the agent of equine granulocytic ehrlichiosis (EGE) of horses, and an agent of canine granulocytic ehrlichiosis in the US, South America, and Europe; E. phagocytophila, the agent of tick-borne fever of ruminants in Europe; an as yet unnamed Ehrlichiae that is the causative agent of human granulocytic ehrlichiosis (HGE) in the United States and Europe; and more distantly, E. platys, a thrombocytic

— o — Ehrlichiae that causes mild cyclical thrombocytopenia in dogs .

Emerging genetic and antigenic data indicates that the members of the E. phagocytophila genogroup are very closely related or identical species [Chen et al, J. Clin. Microbiol. , 32:589 (1994)] . In humans, HGE was first recognized in 1990 (Chen et al, supra) , and is considered an emerging disease of increasing clinical significance. With the recent recognition that the agent of HGE is nearly identical genetically (Chen et al, supra) , and antigenically [Dumler, J. Clin. Microbiol. , 33:1098 (1995)], biologically with E. equi, and is capable of causing severe and fatal human infection, there has been an increased awareness of the prevalence of equine and canine infections.

None of the granulocytic Ehrlichiae have been continuously propagated in vitro. This has continuously hampered the development of diagnostic tools for these infections, and investigation of the diseases and causative agents.

Tetracycline antibiotics are the drug of choice for treatment of all ehrlichioses, and most human patients respond with a dramatic defervescence after therapy [Dumler et al, Clin. Infect. Dis . , 20:1102 (1995)] . In most cases, prevention of ehrlichioses focuses on vector control and prophylaxis using tetracyclines. Serologic evidence of E. equi infection in some regions of California has been identified in up to 50% of the horses residing in those regions (Madigan et al, J. Am. Vet. Med. Assoc, 196:1962 (1990)) . Similarly, nearly 20% of animals tested in a serosurvey of ill dogs in Oklahoma had evidence of E. equi infection (Rodgers et al, J. Vet. Diagn. Invest. , 1:154 (1989) ) .

Since the first identified case of HGE in 1990, there has been a logarithmic increase in the number of diagnoses of that human infection, especially since modern diagnostic methods have become available through specialized academic research facilities and some commercial laboratories. To date, approximately 115 cases of HGE have been recognized in the United States [Wormser et al, MMWR, 44:593 (1995)] . Initial studies suggest that approximately 10% of human patients with Lyme disease may have been infected with the agent of HGE. Given the nearly 10,000 cases of Lyme disease reported annually in the United States, one would speculate that perhaps 1,000 of these patients may have also acquired undiagnosed HGE, in addition to those patients with HGE not accompanied by Lyme disease.

Ehrlichia phagocytophila and E. equi are transmissible through the bite of Ixodes ricinus (MacLeod et al, Parasitology, 25:273 (1933)) and I. pacificus ticks, respectively. Mounting evidence has implicated Ixodes scapularis {dammini) ticks as the vector responsible for transmitting the agent of HGE in the United States (Bakken et al, JAMA, 272:212 (1994) ) . It is thought that larval ticks acquire the pathogen from a reservoir host, probably wild rodents, and that subsequent developmental stages; i.e., the nymphs and adults, transmit the Ehrlichiae during their blood meal .

Specific diagnosis of HGE, EGE, and tick- borne fever is accomplished by serology utilizing antigen prepared from infected animals. Diagnosis in animals is often suspected when typical intracytoplasmic inclusions are present in the peripheral blood leukocytes of febrile animals. In contrast, Ehrlichiae inclusions are variably present in the peripheral blood of humans who nevertheless may be very ill.

It would be beneficial if a definitive and less invasive diagnosis of SLE patients could be found, and if the patients so identified could be more effectively treated. The description that follows describes a diagnostic method that is believed to be definitive for SLE, and a treatment regimen that deals with the causative agent in SLE and can eliminate that agent from a patient's body.

BRIEF SUMMARY OF THE INVENTION The present invention contemplates a method of diagnosing systemic lupus erythematosus (SLE) in a human patient. The method comprises the steps of providing an analyte from the human that may contain a target nucleic acid sequence of SLE-causing infectious bacterium, adding to the analyte at least one nucleic acid probe designed to hybridize to the target nucleic acid sequence to form a mixture. The mixture so formed is maintained (incubated) under conditions permitting formation of a nucleic acid (target/probe) hybrid molecule from the probe nucleic acid and the target nucleic acid sequence if the target nucleic acid sequence is present. The presence or absence of a target/probe hybrid nucleic acid hybrid is assayed determined. Illustrative nucleic acids probes have a sequence selected from the group consisting of SEQ ID NOs: 1-9.

In another aspect of the invention, antibodies from a patient to be diagnosed for SLE are admixed with a promyelocyte Ehrlichiae Ep group antigen, such as that provided by an HL60 cell culture is infected with an Ehrlichia phagocytophiIa genogroup organism. The antigen/antibody admixture is maintained for a time period sufficient to immunoreact and form an immunoreaction product . SLE is diagnosed in the patient by detecting the presence of an immunoreaction product. In one embodiment, the Ehrlichiae Ep group antigen is from Ehrlichia equi . The immunoreaction product can be detected by several well known means such as an indirect immunofluorescence, an indirect immunoenzymatic, latex agglutination, and complement fixation.

The present invention has several benefits and advantages. One benefit of the invention is that an assay provides a definitive diagnosis as compared to a differential diagnosis as is currently the case.

An advantage of the invention is that it provides two independent assay methods that can provides a definitive diagnosis of SLE.

Still further benefits and advantages of the present invention will be apparent to a skilled worker from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS In the drawings forming a part of this invention:

FIG. 1 is taken from the inventor's U.S. Pat. Nos. 5,972,309 and 5,795,563 and shows a thin blood film obtained from a patient of the Examples and stained for one hour with filtered Giemsa stain. This patient was splenectomized and had systemic lupus erythematosus . Several individual erythrocytes are visualized. The stained structure within the erythrocyte indicated by the arrow is the infectious bacterium thought to cause SLE. The structure in another erythrocyte designated "H" is a Howell-Jolly body often seen in splenectomized patients. A Howell-Jolly body can be differentiated from the bacterium by size and phase refraction but not by staining characteristics. Original magnification X630.

FIG. 2 is the same field of erythrocytes of the patient as seen in FIG. 1 and is also taken from the inventor's U.S. Pat. No. 5,972,309 and No. 5,795,563. The same erythrocytes are visualized, but the optical view is by phase contrast. In this mode, the Giemsa-stained bodies are poorly visualized. Doubly retractile structures occupy the same positions and locations within the erythrocytes of those structures designated as the infectious bacterium, as seen with phase contrast optics. The structure of FIG. 1 designated "H" is absent in the phase contrast view as the Howell-Jolly structure does not contain a retractile body. Original magnification is X630.

FIG. 3 is an electron micrograph taken from the inventor's U.S. Pat. Nos . 5,972,309 and 5,795,563 and showing a small portion of an erythrocyte from the same patient as in FIGS. 1 and 2. The area designated "E" is the matrix of the erythrocyte. The small arrow above and to the right of the "E" designates the erythrocyte membrane. The large arrow designates the double membrane enclosing the exogenous, infectious bacterium, and appears to have some of the characteristics of a gram-negative bacterial membrane. The limiting membrane of the vacuole layer surrounding the bacterium is designated by the unmarked wedge near the left-hand side of the micrograph, and morphologically resembles erythrocyte membrane. The original magnification X65,000.

FIG. 4 is a photomicrograph of a megakaryocyte from another SLE patient . The arrow adjacent to "mh" indicates an inclusion morphologically similar to the infectious bacteria seen in FIGS. 1-3. The arrow adjacent to "L" indicates a lymphocyte, whereas the arrow adjacent to the "N" indicates a nucleus.

FIG. 5, is adapted from Iwasaki et al . , Immunology, 19:451-462 (2003), is a schematic depiction of hematopoietic cell differentiation in which HSC=Human Stem Cell, CMP=myelomocytic progenitor, CLP=Lymphoid progenitor, GMP=granulocyte- monocyte progenitor, MEP=erythrocyte-megakaryocyte progenitor, and ProT=Lymphocyte T cell progenitor, and ProB=lymphocyte B cell progenitor.

Definitions

As used herein, the 3 '-terminal region of the nucleic acid probe refers to the region of the probe including nucleotides within about 10 residues from the 3 ^■ -terminal position.

In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or "5¹" relative to an element if they are bonded or would be bonded to the 5 ' -end of that element. Similarly, discrete elements are "downstream" or "3¹" relative to an element if they are or would be bonded to the 3 '-end of that element. Transcription proceeds in a 5 ' to 3 ' manner along the DNA strand. This means that RNA is made by the sequential addition of ribonucleotide-5 ' - triphosphates to the 3 ' -terminus of the growing chain (with the elimination of pyrophosphate) .

As used herein, the term "target nucleic acid" or "nucleic acid target" refers to a particular nucleic acid sequence of interest. Thus, the "target" can exist in the presence of other nucleic acid molecules or within a larger nucleic acid molecule .

As used herein, the term "nucleic acid probe" refers to an oligonucleotide or polynucleotide that is capable of hybridizing to another nucleic acid of interest. A nucleic acid probe may occur naturally as in a purified restriction digest or be produced synthetically, recombinantly or by PCR amplification. As used herein, the term "nucleic acid probe" refers to the oligonucleotide or polynucleotide used in a method of the present invention. That same oligonucleotide could also be used, for example, in a PCR method as a primer for polymerization, but as used herein, that oligonucleotide would then be usually referred to as a "primer" . Herein, oligonucleotides or polynucleotides may contain a phosphorothioate bond.

As used herein, the terms "complementary" or "complementarity" are used in reference to nucleic acids (i.e., a sequence of nucleotides) related by the well-known base-pairing rules that A pairs with T and C pairs with G. For example, the sequence 5 ' -A- G-T-3¹, is complementary to the sequence 3'-T-C-A-5' . Complementarity can be "partial," in which only some of the nucleic acid bases are matched according to the base pairing rules. On the other hand, there may be "complete" or "total" complementarity between the nucleic acid strands when all of the bases are matched according to base pairing rules . The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands as known well in the art. This is of particular importance in detection methods that depend upon binding between nucleic acids, such as those of the invention. The term "substantially complementary" refers to any probe that can hybridize to either or both strands of the target nucleic acid sequence under conditions of low stringency as described below or, preferably, in polymerase reaction buffer (e.g., Promega, M195A) heated to 95°C and then cooled to room temperature. As used herein, when the nucleic acid probe is referred to as partially or totally complementary to the target nucleic acid, that refers to the 3 ' -terminal region of the probe (i.e. within about 10 nucleotides of the 3 '-terminal nucleotide position) .

As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acid strands. Hybridization and the strength of hybridization (i.e., the strength of the association between nucleic acid strands) is impacted by many factors well known in the art including the degree of complementarity between the nucleic acids, stringency of the conditions involved affected by such conditions as the concentration of salts, the T_m (melting temperature) of the formed hybrid, the presence of other components (e.g., the presence or absence of polyethylene glycol) , the molarity of the hybridizing strands and the G: C content of the nucleic acid strands. As used herein, the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds, under which nucleic acid hybridizations are conducted. With "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of "weak" or "low" stringency are often required when it is desired that nucleic acids which are not completely complementary to one another be hybridized or annealed together. The art knows well that numerous equivalent conditions can be employed to comprise low stringency conditions. However, illustrative high, moderate and low stringency conditions are set out below.

Polynucleotide hybridization is a function of sequence identity (homology) , G+C content of the sequence, buffer salt content, sequence length and duplex melt temperature (T_m) among other variables. See, Maniatis et al . , Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1982) , page 388.

With similar sequence lengths, the buffer salt concentration and temperature provide useful variables for assessing sequence identity (homology) by hybridization techniques. For example, where there is at least 90 percent homology, hybridization is carried out at 68°C in a buffer salt such as 6XSCC diluted from 20XSSC [Maniatis et al., above, at page 447] and two sequences form a hybrid duplex (hybridize) . The buffer salt utilized for final Southern blot washes can be used at a low concentration, e.g., 0. IXSSC and at a relatively high temperature, e.g. 68⁰C. Use of the above hybridization and washing conditions together are defined as conditions of high stringency or highly stringent conditions.

Moderately high stringency conditions can be utilized for hybridization where two sequences share at least about 80 percent homology. Here, hybridization is carried out using 6XSSC at a temperature of about 50-55⁰C. A final wash salt concentration of about 1-3XSSC and at a temperature of about 60-68⁰C are used. These hybridization and washing conditions define moderately high stringency conditions .

Low stringency conditions can be utilized for hybridization where two sequences share at least 30 and more preferably about at least 40 percent homology. Here, hybridization carried out using 6XSSC at a temperature of about 40-50⁰C, and a final wash buffer salt concentration of about 6XSSC used at a temperature of about 40-60⁰C effect non-random hybridization. These hybridization and washing conditions define low stringency conditions.

As used herein, the term "T_m" is used in reference to the "melting temperature" . The melting temperature is the temperature at which 50% of a population of double-stranded nucleic acid molecules becomes dissociated into single strands. The equation for calculating the T_m of nucleic acids is well-known in the art. The T_n, of a hybrid nucleic acid is often estimated using a formula adopted from hybridization assays in 1 M salt, and commonly used for calculating T_m for PCR primers: {[(number of A+T) X2°C + (number of G+C) X 4°C] } Newton et al . PCR, 2.sup.nd Ed., Springer-Verlag (New York: 1997), p. 24. This formula was found to be inaccurate for primers longer that 20 nucleotides. Id. Other more sophisticated computations exist in the art that take structural as well as sequence characteristics into account for the calculation of T_m. A calculated T_n, value is merely an estimate; the optimum temperature is commonly determined empirically.

The term "homology, " as used herein, refers to a degree of complementarity. There can be partial homology or complete homology (i.e., identity) . A partially complementary sequence that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous . "

When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous," as used herein, refers to a probe that can hybridize to a strand of the double-stranded nucleic acid sequence under conditions of low stringency.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the detection of an SLE-causing infectious bacterium in a human body sample and tracking of the treatment of systemic lupus erythematosus in a human.

Thus, one aspect of the method comprises determining the presence in that human body sample of a sequence of the nucleic acid that encodes the SLE- causing infectious bacterium. A contemplated human body sample preferably contains blood cells such as those shown and enumerated in Fig . 5 , and can illustratively be whole blood, red blood cells, marrow, neutrophils, white cells and blood stem cells. These cells provide a nucleic acid-containing analyte that is assayed. Preferably, that bacterial nucleic acid hybridizes with one or more of SEQ ID Nos:l-9. Those hybridizations are typically carried out under conditions of moderate to high stringency as are discussed hereinafter.

The SLE-causing infectious bacterium is found by polymerase chain reaction (PCR) from a splenectomized human patient who had 3.8 percent of her erythrocytes infected with the organism similar to the animal hemotropic mycoplasmas, the Ehrlichia equi Ep group, Anaplasmataceae and Haemobartonella- like organisms described before. The new organism is thought closest to the Ehrlichia equi Ep group.

The SLE-causing infectious bacterium infection present in vivo can be detected in vitro by PCR using primers designed specifically to amplify only Ep group DNA as well as with SEQ ID NO: 4. PCR can be conducted on isolated infected cell culture DNA.

Primers are designed based upon the DNA sequence of the 16S ribosomal RNA gene in regions conserved among E. equi, E. phagocytophiIa, and the HGE agent. Primers are typically complementary to and/or hybridize to a 5 ' region and a 3 ' region of the nucleic acid sequence. Primers preferably have at least 15 nucleotides that are complementary to the nucleic acid sequence. Primers are preferably about 15 to about 50 nucleotides long and can be prepared by automated synthesis.

Two sets of preferred useful primers (SLEl- F, SLEl-R, SLE2 and SLE3) amplify a 919 base pair fragment of the Ep group 16S ribosomal RNA gene and have the following sequences [Chen et al, J. Clin.

Microbiol. , 32:589 (1994)] and US Patent No.

5,955,359:

SLEl-f 5'-AACGGATTATTCTTTATAGCTTGCT-B' (SEQ ID NO: 1)

SLEl -r 5' -GGAGATTAGATCCTTCTTAACGGAA-B' (SEQ ID NO: 2); or a 928 base pair fragment of the Ep group 16S ribosoraal RNA gene and have the following sequences:

SLE2 5'-GTCGAACGGATTATTCTTTATAGCTTGC-B' (SEQ ID

NO : 3 ) , SLE3 5 ' -CCCTTCCGTTAAGAAGGATCTAATCTCC-3 ^■ (SEQ

ID NO: 4) .

A further useful sequence obtained from an SLE patient blood is designated SLE4 and has the sequence: 5'-TCGTAACCAA TCTCAAGCTC AACCCTGGCA CCACCAATAC CATAACCAAC ACTGCCTTCC ATAGCTACAA GCATGTTGTC CTTAAACCCA ATCCGAGGAT CAGGAGTGTT CCAGTCA (SEQ ID NO: 5) and can be used in hybridization assays .

Alternately, the following primers can be constructed to specifically amplify a 150 bp DNA fragment from E. equi and Ep group Ehrlichiae as discussed for other purposes in US Patent No. 5,928,879. Examples of such primers include: SLE5 5'-TAGATCCTTCTTAACGGAAGGGCG-B' (SEQ ID NO: 6); SLE6 5 < -AAGTGCCCGGCTTAACCCGCTGGC-3 ' (SEQ ID NO: 7); SLE7 5'-TTTATCGCTATTAGATGAGCCTATG-B' (SEQ ID NO: 8); and SLE8 5' -CTCTACACTAGGAATTCCGCTAT-S' (SEQ ID NO: 9)

The present invention also contemplates a method for diagnosis of systemic lupus erythematosus in a human that utilizes the human' s antibodies as a body sample and an antigen from a promyelocyte cell preparation comprising Ehrlichiae Ep group-infected promyelocyte cell culture, herein referred to as a promyelocyte Ehrlichiae Ep group antigen. The human's antibodies are admixed with a promyelocyte Ehrlichiae Ep group antigen to form an antigen/antibody admixture. The antigen/antibody admixture is maintained for a time period sufficient to immunoreact and form an immunoreaction product . SLE is diagnosed in the patient by detecting the presence of an immunoreaction product .

Illustrative promyelocyte cells include HL60 and KG-I human cell lines, which cell lines were deposited with the ATCC in 1982 (ATCC No. CCL 240 and ATCC No. CCL 246) . Additional promyelocytic leukemia cell lines that can also be used include K-562 (ATCC No. CCL 243), HEL-92.1.7 (ATCC NO. TIB 180), ML-I

[Kastan et al., Cancer Res., 51:6304-6311 (1991)] , X-CGD PLB-985 [Ding et al . , Blood, 88:1834-1840

(1996)] , LAMA-84 (Blom et al . , Scand. J. Immunol. , 44:54-61 (1996)] , M-NF5-60 (ATCC No. CRL 1838), WEHI- 3 (ATCC No. TIB 68), and Ml (ATCC No. TIB 192) . HL60 and KG-I cell cultures are preferred.

Antigens for diagnostic serologic tests are preferably prepared by harvesting sufficient quantities of infected HL60 or KG-I cells when approximately 50% or more of the cultured cells contain Ep group Ehrlichiae. The antigens can be used directly as fixed whole infected cells for indirect fluorescent antibody tests or can be purified by density-gradient centrifugation of lysed, infected tick cell cultures . The antigens so prepared can then be admixed with serum from a person to be diagnosed for the presence or absence of SLE to form the antigen/antibody immunoreaction product. The presence or absence of that immunoreaction product and thereby the presence or absence of SLE can be determined using latex agglutination, indirect immunofluorescence, ELISA, solid phase enzyme immunoassay, indirect immunoenzymatic assay, complement fixation, immunoblot, or the like.

Nucleic acids derived from the Ehrlichiae cultured in HL60 and KG-I cells can be used to prepare recombinant bacteriophage, viral, cosmid, phagemid, or plasmid genes that when transfected into appropriate hosts (bacteria, yeast, eukaryotic cells) express recombinant Ep group Ehrlichial proteins useful as diagnostic reagents

EXAMPLES

The following examples are presented to illustrate certain aspects of the invention and are not intended to limit the scope of the invention.

Example 1 : Human Patients

Patient A

Patient A, a 36 -year-old African-American female was hospitalized in May 1988 for treatment of idiopathic thrombocytopenic purpura, caused by presumed hypersplenism. She underwent splenectomy and within three years had developed the multiple laboratory and clinical abnormities that were defining of SLE. These abnormalities consisted in part of elevated anti-dsDNA, arthritis, cutaneous lesions consistent with lupus, and lupus nephritis. When examined in 1994, her erythrocytes had intra- erythrocytic inclusions occupying 16% of the counted erythrocytes. These Giemsa-stained particles were about 0.5 μ in diameter, inside the erythrocyte in the marginal position, and did not appear to cause changes in the erythrocytic structure. By bright field light microscopy they had no internal structure, and, because they appeared to be contained within a vacuole, were different in appearance from Howell-Jolly bodies when visualized by phase microscopy.

A trial of doxycycline 100 mg twice daily was instituted, based on known activity against erythrocyte-associated-organisms erythrocyte- associated organisms in related erythrocyte animal infections such as bovine anaplasmosis [Wanduragala et al . , "Anaplasmosis" in Rickettsial and Chamydial Diseases of Domestic Animals, Woldehiwet et al . eds . , Oxford, Pergamon Press, Chapter 3, 69-88 (1993)] . This treatment was followed with treatment by clarithromycin 250 mg q 12 hours, and rifampicin 600 mg daily. During the period of antibiotic treatment in 1996, she was evaluated monthly with counts of erythrocyte parasitization, studies of anti dsDNA, and general medical physical examination including evaluation of her oral and cutaneous lesions.

Patient B

Patient B was a Caucasian female born in 1976, had never left Norway, and first had thrombocytopenia at age 14. She underwent splenectomy at age 15 and following this procedure, over the next two years, experienced a cascade of thrombocytosis, butterfly malar rash, a positive anti -nuclear antibodies (ANA) , and arthralgia. A kidney biopsy was consistent with lupus nephritis. These findings were consistent with the diagnosis of SLE. She subsequently suffered several episodes of thromboembolic disease, which resulted in loss of the left leg, left cerebral stroke with right hemi- paresis, and an intra-cardiac thrombus by age 19. Following discovery of intra-erythrocytic structures, she was treated with doxycycline 100 mg twice daily, and following this, with clarithromycin 250 mg q 12 hours, and rifampicin 600 mg daily. All antibiotics were discontinued by her medical attendants after the thrombus to her leg. She suffered a myocardial infarction at age 21.

Her peripheral blood smear was examined on three occasions in 1995, and twice in 2003. The percentage of erythrocytes with inclusions observed optically was as follows: 1995, 0.5%; 2003, 3.8% and 0.6% six months later.

Determination of Percentage of Affected

Erythrocytes

Blood smears collected for examination were spread as thin smears on pre-cleaned glass microscope slides and fixed in absolute methanol. On an optical projection of appropriate stained thin sectors of blood with observations only of erythrocytes, a count is taken of all the erythrocytes seen within the borders of the optical projection with the infected erythrocytes noted, counted, and the result calculated and expressed as the percent parasitized erythrocytes .

Blood Studies

For use in whole blood electron microscopy, heparinized blood was washed with 20Xvolumes of PBS pH 7.4, and centrifuged. Cells in the resulting pellet were resuspended in freshly prepared paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4. The initial fixation was carried out at 4°C for one hour. Fixed cells were then washed twice in 0.1 M cacodylate buffer and resuspended in 2% low temperature gelling agar. Then, 1-1.5 mm diameter ribbons of agarose containing fixed cells were fixed in the original fixative for two hours at 4°C. Ribbons were washed several times and post fixed with 2% osmium tetroxide in cacodylate buffer for one hour on ice. Finally, these fixed ribbons were washed several times with water, dehydrated with successively increasing concentrations of alcohol, immersed in two changes of propylene oxide, then immersed in propylene oxide/Spurr ' s resin (1:1 mix) overnight prior to embedding in Spurr's resin and polymerization of blocs in 60⁰C oven.

In other blood studies, blood smears were prepared on clean glass slides, air-dried and fixed in methanol for 2 minutes . Stock Giemsa stain was diluted 1:10 with buffer solution. The working stain solution was filtered through a 0.2 micron pore filter, and overlaid directly on the smear for one hour.

Slides were examined at 500X and IOOOX magnification. When a representative intra- erythrocytic structure was seen with transmitted bright field illumination, the optics were changed to phase contrast, without moving the slide. The blue gray erythrocytic inclusions were interpreted as enclosed win a vacuole

In appropriately spread areas of Giemsa- stained peripheral blood smears, all erythrocytes and infected erythrocytes were counted. The percentage of infected erythrocytes was calculated.

In examinations for mitochondria, blood smears were subjected to staining and fluorescent spectroscopy. The thin blood smears were fixed in absolute methanol, overlaid with MitoTracker.RTM. (MitoTracker.RTM. Green FM, Invitrogen, Carlsbad, Calif.) and examined by epifluorescent microscopy. When a fluorescent intra-erythrocytic structure was found, the optics were switched to phase contrast to further determine if the structure were coincident with another inclusion.

Examination of Archived Bone Marrow Specimens

Twenty specimens of bone marrow, taken from archives at Rush University Medical Center, were numbered. These specimens included bone marrow specimens from 14 patients with SLE and 20 patients without that diagnosis. This information had been extracted from their record by a third party and was kept confidential to blind the specimens to the investigators .

The blinded specimens were examined by two investigators at 500 to 1000 X, with particular attention to the megakaryocytes (MGKC) with at least 20 megakaryocytes visualized. If intercalating- dyable intra-megakaryocytic inclusions were seen, the marrow was declared to be positive. If fewer than 20 MGKC were seen, the specimen was not included in the results. If too much stain precipitate was present, the specimen was not counted. When the code was broken, the identifying number was removed, and replaced by a study designation, so that the identity of any patient was not present in any interpretation.

Results of Antibiotic Treatment- -General

Clinical parameters of objective and subjective disease of both lupus patients A and B improved with both doxycycline and clarithromycin and rifampicin, though the improvement of subjective symptoms seemed greater with the latter combination to the patients as well as the investigators. Both patients had changes in their erythrocytes following clarithromycin and rifampin that suggested bacterial disintegration and disappearance of bacterial substance within the vacuoles . Multiple vacuoles post bactericidal antibiotics were also seen without phase illumination suggesting emptying of the contents of the vacuoles . These empty vacuoles seen under bright field illumination were not present in either patient or any other patient's blood examined before the antibiotic therapy though all specimens were collected and treated in the same way.

Results of Antibiotic Therapy

Patient A

Upon institution of therapy with doxycycline, the percentage of parasitized erythrocytes began a decline over several months. This decline appeared to be logarithmic, and was confirmed by a graph of the logs of the percentage of parasitized erythrocytes, which was linear. This decline was accompanied with clinical improvement in skin lesions and oral ulcerations as well as a decline in anti dsDNA levels after treatment with Doxycycline for 6 months. However, by bright field microscopy in Giemsa- stained blood films, there were still intra-erythrocytic inclusions that were enclosed in a vacuole, suggesting that although significant clinical improvement occurred as well as a decline in anti-dsDNA, and a decline in intra- erythrocytic bodies, it appeared that doxycycline had not completely eliminated the inclusions.

The second antibiotic course of clarithromycin and rifampicin caused a fundamental change in the inclusions. Within several weeks, the intra-erythrocytic structures became fragmented and disappeared from the vacuoles in the erythrocytes. The vacuoles could now be seen without phase contrast optics and continued clinical improvement occurred. The cutaneous lesions on the neck and face continued their resolution, and the oral lesions disappeared. The patient returned to treatment by a rheumatologist and was lost to follow-up.

Patient B

Reports of the patient condition suggested marked clinical improvement with antibiotic therapy, and after the treatment with clarithromycin plus rifampicin was begun, similar changes in the intra- vacuolar bodies to those seen in patient A occurred, with fragmentation and the appearance of empty vacuoles in the erythrocyte following the bactericidal antibiotics. All antibiotics were discontinued by the treating physician following severe thromboembolic events coincident with extremely high platelet counts. Her peripheral blood smear was examined on three occasions: once in 1995 and twice in 2003. The empty vacuoles seen under bright field illumination were not seen in any other patients, although all specimens were treated in the same way.

Observations of Giemsa-Stained Blood Films of Splenectomized Lupus Patients

Seven splenectomized lupus patients were studied, including patients A and B. All upon careful examination had 0.3-0.5 micron in diameter intra-erythrocytic bodies, which upon phase contrast optics appeared to be within a vacuole. With use of MitoTracker^® and phase contrast microscopy, showed the intra-vacuole structures to be different from mitochondria. No percentage of erythrocytes with inclusions exceeded 1%, within parameters permitted by sampling error. The two splenectomized index patients had from about 4% (Patient B) to about 16% (Patient A) of the observed erythrocytes parasitized.

Blood from Patient B was observed in 1995 before antibiotic treatment and less than 1% of the erythrocytes exhibited inclusions. When her blood was examined in 2003, and she was under no antibiotic treatment, the count was about 4%. The DNA extracted from this specimen led to the determination of a mycoplasmal 16 S rRNA by PCR. Six months later, the percentage of infected erythrocytes had returned to less than 1%.

Electron Microscopy of Contents of an Intra- Erythroctic Vacuole in Patient A

Intra-erythrocytic vacuoles tended to be oval in shape, and were often paired with one or more bacteria-like structures within one or both of the paired structures. A single vacuole was closely examined. The bacteria like structure was 0.5 micron in diameter, and was enclosed within a vacuole that was similar in appearance to the erythrocyte limiting membrane. Observable internal features included two well-defined structures that resembled tangential sections of a helical cytoskeleton. There was no rigid cell wall. Multiple observations of this fixed block of blood, sampled at approximately 8% parasitemia, confirmed that the structures seen were repeatedly observed, and confirmed the level of parasitemia observed with light microscopy when the blood was originally drawn. This blood sample was taken during the course of therapy with doxycycline, and the percentage had declined from about 16% to about 8%. The structures were consistent with a member of the Mollicutes, because members of the Mollicutes, Spiroplasma, Acholeplasma, and Mycoplasma, exhibit a cytoskeletal structure. [Razin et al . , Microbiology and Microbiological Reviews, 62:108-2172 (1992) .] The intra-bacterial structures were different from mitochondrial cristae, which are contiguous with the cell membrane.

Microscopic Examiniation of Archived Bone Marrow Specimens

A search of megakaryocytes in lupus marrow specimens was conducted after marrow of a patient with SLE and persistent leucopenia was examined, and an inclusion identified. See, FIG. 4.

A series of marrow aspirate slides was examined with the identity of the patient ' s disease being blinded to the examiner. After the codes were broken, fourteen specimens were found to have been evaluated from patients with a diagnosis of SLE: 11 of 14 of those patients had inclusions in their megakaryocytes consistent with small bacterial structures as seen by Giemsa-based stain. Of twenty patients with preliminary diagnoses other than lupus, nine had inclusions consistent with the described intra-megakaryocytic structures identified by morphology of small inclusions near the limits of optical resolution, and complicated by granules from disrupted cells in marrow aspirates. On re-examination of marrow slides by the investigators, the proportion of lupus marrows with megakaryocytic inclusions remained significantly higher than "controls", but the concordance of results on specific specimens was not exact. It was concluded that differences in results from various readings of the still blinded specimens were attributable to an attempt to identify morphology of small inclusions near the limits of the optical resolution, and complicated by granules from disrupted cells in marrow aspirates.

Example 2 : Preparation of Ehrlichia-infected Cells Following the teachings of Dumler et al . , US Patent No. 5,955,359, Ep group granulocytic Ehrlichiae such as Ehrlichia equi or the human granulocytic Ehrlichia obtained from infected horses or the human granulocytic Ehrlichia obtained from human patients with human granulocytic ehrlichiosis can be grown in promyelocyte cell cultures. Thus, Ep group granulocytic Ehrlichiae, such as E. equi or the human granulocytic Ehrlichia, are incubated with promyelocyte cell line cultures, preferably HL60 or KG-I cell cultures. The E. equi and human granulocytic Ehrlichia inoculum can be obtained from horses after experimental inoculation with thawed, E. equi- or human granulocytic Ehrlichia-infected blood from an acutely infected horse, followed by venipuncture and removal of fresh, infected blood at a time when Ehrlichiae are visible in the peripheral blood leukocytes of the ill, infected horse. Alternately, the human granulocytic Ehrlichia inoculum can be obtained from human patients during the acute phase of human granulocytic ehrlichiosis at a time when typical Ehrlichia morulae are detected within leukocytes in the peripheral blood of the infected patient.

In accordance with Dumler et al . US Patent No. 5,928,879, Ehrlichia equi obtained from infected horses can be grown in I. scapularis tick cell culture. In a method of that patent, Ep group granulocytic Ehrlichiae, such as E. equi, are incubated with I. scapularis tick cell culture, preferable the IDE8 line [deposited with the American Type Culture Collection in Rockville, MD on August 26, 1995, under ATCC No. CRL 11973] .

Tick cell cultures in which about 10% to about 50% of the cells are infected can be mixed with uninfected tick cells at a ratio of about 1:3 to about 1:20. Ehrlichia equi is cultured in I. scapularis cells derived from a suspected natural vector of the pathogen. The requirements for successful isolation of the Ehrlichiae in tick cell culture are: an atmosphere that is reduced in O₂ tension (from 20% O₂ as found in normal air to about 17%) , and enhanced 10-fold with respect to CO₂ tension [from 0.03% as found in normal air to about 3%) . The culture medium used also must include CO₂ in the form of NaHCO₃ (0.25% (w/v) ] , and an organic buffer. An organic buffer, such as 3- (N-morpholino) -propane sulfonic acid (MOPS) or N- (2-hydroxyethyl) piperazine-N- (2-ethanesulfonic acid) (HEPES), at a concentration of 10 to 15 mM is preferably included to ensure growth of E. equi. The infected tick cultures are incubated at about 31°C to about 35°C until growth of E. equi is detected. Ehrlichia equi grows to high yield in IDE8 cells as compared to neutrophilic granulocytes harvested from horses. Individual cells can contain enormous masses (>l,000) of Ehrlichiae.

The organisms found in the neutrophils are contained within a membrane bound vacuole up to about 3 to 5 (Jin in diameter. The individual organisms are small, highly pleomorphic, and approximately 0.5 μm in size. With Romanowsky stains, such as Giemsa or LeukoStat, Ehrlichiae species and the morulae appear bluish-purple. By electron microscopy their electron density is similar to that of the host cytoplasm, but some morulae may contain smaller, dense forms.

Illustrtatively, Ep group granulocytic Ehrlichiae, such as E. equi or the human granulocytic Ehrlichia agent, in acute phase blood samples of naturally infected humans or animals or experimentally- infected animals such as horses, are Co- incubated with log phase growth of promyelocytic leukemia cells, such as HL60 or KG-I cells.

Illustratively, KG-I cells are preferably cultured in suspension in a tissue culture medium, such as RPMI 1640 supplemented with 5.0 to 20% (v/v) , preferably about 10% (v/v) , heat-inactivated fetal bovine serum and 2.0 mM L-glutamine and in an atmosphere of 5% CO₂ at about 37°C. Antibiotics, such as penicillin with streptomycin, or gentamicin can be added for maintenance cultures to suppress bacterial contamination, as is standard in the art. The doubling time of the cultures under these conditions is approximately 48 to 72 hours, and thus cell concentration should be carefully maintained, preferably below 1.0X10⁷ cells per ml of tissue culture medium. Thus, log phase growth HL60 or KG-I cells are centrifuged and resuspended in tissue culture medium at a concentration of about 3. OXlO⁶ cells per ml. Cell culture medium is preferably partially replaced 2 to 3 times per week with freshly prepared medium.

The HL60 (ATCC No. CCL 240) and KG-I (ATCC No. CCL 246) human promyelocytic leukemia cell lines are propagated in RPMI 1640 medium with 10 to 20% (v/v) fetal bovine serum, 2.0 mM L-glutamine, with or without penicillin and streptomycin. Log phase HL60 or KG-I cells are centrifuged, counted, and resuspended into fresh medium without penicillin and streptomycin at a cell density of about 3. OXlO⁶ cells per ml. Whole ethylene diaminotetraacetic acid (EDTA) or acid citrate dextrose (ACD) anticoagulated human or equid blood known to be infected with the human granulocytic Ehrlichia or E. equi, of which less than 10% of human peripheral blood neutrophils contained Ehrlichia morulae and less than 50% of equid peripheral blood neutrophils contained E. equi or human granulocytic Ehrlichia morulae, are used.

Between 300 to 500 1 of this infected blood are added directly into 25 cm² plastic tissue culture flasks containing 5.0 to 9.0 ml of HL60 or KG-I cells. Similarly, an equivalent inoculum of uninfected human or equid blood, or blood from a patient recovering from human granulocytic ehrlichiosis after 2 to 3 days of doxycycline therapy is incubated with 5.0 to 9.0 ml of HL60 or KG-I cells, and 5.0 to 9.0 ml volumes of HL60 cells are held as uninoculated controls. The flasks are maintained in an atmosphere of 5% CO₂ at 37°C.

Cell cultures are examined every two to four days by removing small aliquots and preparing cytocentrifuged slides that were then examined microscopically after Romanowsky (LeukoStat, Fisher Scientific, Pittsburgh, PA) staining. Aliquots of the same cells are stored for later examination to detect the presence of Ehrlichiae species and Ep group granulocytic Ehrlichiae DNA.

Before day 3, control HL60 and KG-I cells, control HL60 and KG-I cells inoculated with uninfected blood, blood from a doxycycline-treated patient recovering from human granulocytic ehrlichiosis, and HL60 cells inoculated with E. equi- infected equid blood appear identical, except for the presence of occasional normal blood erythrocytes and leukocytes in the blood-inoculated cultures among the HL60 or KG-I leukemia cells. By 3 days after inoculation, typical intracytoplasmic inclusions (morulae) filled with individual bacterial bodies {Ehrlichiae) are present within vacuoles of 3.0% of cells in the flask of KG-I cells inoculated with human granulocytic Ehrlichia- infected equid blood. By 4 days after inoculation, typical intracytoplasmic inclusions (morulae) filled with individual bacterial bodies {Ehrlichiae) are present within vacuoles of 12.0% of cells in the flask of HL60 cells inoculated with E. equi-infected equid blood, and by 5 to 9 days after inoculation, typical intracytoplasmic inclusions (morulae) filled with individual bacterial bodies {Ehrlichiae) are present within vacuoles of 21.0 to 31.0% of cells in the flask of HL60 cells inoculated with human granulocytic Ehrlichia- infected human blood.

No morulae are noted in any of the control HL60 or KG-I flasks. Between 2 to 3 weeks after inoculation of cultures with infected blood, morulae are present in from zero to 100% of cells, and in many cases, multiple, complex morulae in various stages from discrete organisms to large aggregates, and in some instances appearing to cause cell lysis and subsequent extracellular release of organisms. Control cells contain no such structures. The morphology of many of these morulae, especially in the early cultures, is quite similar to those seen in the peripheral blood neutrophils in human granulocytic ehrlichiosis in that these appear to have generally even, rounded contours within the apparent cytoplasmic vacuoles .

The Co-cultivated cells are maintained in suspension cultures and are examined periodically, preferably weekly, by Romanowsky staining or immunofluorescence, for the presence of typical Ep genogroup Ehrlichia species morulae and antigens indicative of infection. After about ten days, morulae are detectable by Romanowsky staining, and by day 21, more than 50% of the HL60 cells contain Ehrlichia species morulae. The infected HL60 or KG-I cells can then be harvested and used for antigen or nucleic acid preparation.

The above -prepared E. equi-infected IDE8 cells can also be used to infect human promyelocytic leukemia cell lines. For example, E. equi- infected IDE8 cells are mixed with the HL60 cells, preferably at a ratio of approximately 1:36 (0.25 ml of infected tick cells and 9.0 ml of HL60 cells) . The preferred culture conditions include RPMI 1640 medium supplemented with about 10% (v/v) fetal bovine serum (heat-inactivated), about 2.0 mM L-glutamine, and in an atmosphere of 5% CO₂ at about 37°C. The co- cultivated cells and HL60 cells are maintained in suspension cultures and are examined periodically, preferably weekly, by Romanowsky staining, for the presence of typical Ehrlichiae morulae indicative of infection. Infected cells can contain single morulae typical in appearance, as seen in the peripheral blood neutrophils of patients with human granulocytic ehrlichiosis, and horses or dogs with E. equi granulocytic ehrlichiosis. With progressive time, an increasing percentage of cells become infected, and may contain multiple morulae, providing an in vitro method to obtain substantial quantities of Ep group Ehrlichiae propagated within mammalian (human) cells.

For assay purposes, it is preferred that about 50-100% of the HL60 cells are infected with Ehrlichia phagocytophilia or Ehrlichia equi. Illustrative immunoassays are often carried out on a microscope slide. Here, about 100 to about 1000 HL60 or other promyelocytic cells are present on a slide, and no less than about 20% of the cells are infected with Ehrlichia phagocytophilia or equi.

Infected cells can contain single morulae typical in appearance, as seen in the peripheral blood neutrophils of patients with human granulocytic ehrlichiosis, and horses or dogs with E. equi granulocytic ehrlichiosis. With progressive time, an increasing percentage of cells become infected, and can contain multiple morulae, providing an in vitro method to obtain substantial quantities of Ep group Ehrlichiae propagated within mammalian (human) cells.

Growth and development of E. equi or the human granulocytic Ehrlichia agent in, e.g., HL60 or KG-I cells can be detected by several methods. One method is to prepare cell spreads using a cytocentrifuge and to stain the cells with a Romanowsky stain. Infected cells can then be detected by the presence of Ehrlichiae inclusions in the cytoplasm. The inclusions seen in HL60 and KG-I cells are similar to those seen in vivo in the neutrophilic granulocytes of humans, dogs and horses. Inclusions in heavily infected cells can completely fill the cytoplasm, and cause the cell to distend and rupture .

Alternatively, growth can be detected by immunocytological methods using specific E. equi, E. phagocytophila, or HGE agent antibodies in indirect fluorescent antibody methods and indirect immunoenzymatic methods.

Promyelocytic leukemia cells, such as HL60 and KG-I cells, infected with E. equi or the human granulocytic Ehrlichia agent or cell cultures are also useful as diagnostic tools for assays including ELISA, indirect fluorescent antibody tests, latex agglutination tests, complement fixation tests, and immunoblot tests. For immunofluorescence, it is preferred that 100 to 1000 cells be present on a slide with about 50% to 100%, and no less than 25% of cells infected.

Example 3 : Illustrative PCR

The identity of E. equi is confirmed using a DNA oligonucleotide primers SLEl-F (SEQ ID NO:1); and SLEl-R (SEQ ID N0:2), by a standard PCR. The antigenic identity of the E. equi in IDE8 tick cultures is also confirmed by an immunocytology using polyclonal horse anti-E. equi and polyclonal human anti-human granulocytic ehrlichiosis agent antibodies .

PCR using infected tick cell culture extract as a template confirms the identity of the E. equi growing in IDE8 cells. A crude lysate is made according to rapid sample preparation for PCR [Higuchi, In: PCR Technology, Principles and Applications for DNA Amplification, H. A. Ehrlich, Ed. Stockton Press, New York, Chapter 4 (1989)] .

Briefly, infected tick cells from one culture are forced about 10 times through a 27 gauge needle, and large debris removed by centrifugation at 10OXg. The supernatant fluid containing small particles and Ehrlichiae is collected by centrifugation at 10,000X.g for 20 minutes, and the pellet is resuspended in lysis buffer with NP-40, Tween^®-20 and Proteinase K (Higuchi, supra) . Following incubation (maintenance) at 55°C for 1 hour, the proteinase is inactivated (95°C for 20 minutes) , and the lysate stored at -20⁰C. Uninfected IDE8 cells are extracted the same way as a control.

Ten μl of that lysate are used as a template in the PCR with primers SLEl-F and SLEl-R at a concentration of 0.5 μM each. 100 μl reaction mixtures containing 1.5 mM MgCl₂ and 0.2 mM of the four deoxynucleotides are cycled 30 times through 92°C for 1 minute, 56°C for 0.5 minutes and 72°C for 1 minute. Ten μl of the resulting DNA is mixed with loading buffer (Ficoll^® 400 with bromophenol blue) , and electrophoresed through 0.9% (w/v) agarose in 0.5X TBE (Tris-Borate-EDTA) buffer at 150 volts until adequately separated.

The gel is stained with ethidium bromide and photographed under UV light. Lysate from infected IDE8 cultures gives rise to a DNA product of the expected size, 919 bp, whereas control lysate does not, indicating the presence of E. equi DNA and thus, E. equi in the infected cultures. A plasmid encoding the entire E. equi 16S ribosomal RNA gene is used as positive control and produces an identical 919 bp band, whereas a negative control using no template produces no detectable band.

Each of the patents and articles cited herein is incorporated by reference. The use of the article "a" or "an" is intended to include one or more .

The foregoing description and the examples are intended as illustrative and are not to be taken as limiting. Still other variations within the spirit and scope of this invention are possible and will readily present themselves to those skilled in the art.

Claims

1. A method for diagnosing systemic lupus erythematosus (SLE) in a human which comprises the steps of:

(a) providing an analyte from said human that may contain a target nucleic acid sequence of SLE-causing infectious bacterium;

(b) admixing with said analyte at least one nucleic acid probe designed to hybridize to the target nucleic acid sequence to form an admixture;

(c) maintaining said admixture so formed under conditions permitting formation of a nucleic acid hybrid molecule from the probe nucleic acid and the target nucleic acid sequence (target/probe hybrid) if the target nucleic acid sequence is present; and

(d) determining the presence or absence of a target/probe hybrid nucleic acid hybrid, the presence of a target/probe hybrid indicating the presence of SLE.

2. The method according to claim 1, wherein said probe nucleic acid has a sequence selected from the group consisting of SEQ ID NOs: 1- 9.

3. The method according to claim 1, wherein said probe nucleic acid has a sequence that includes a sequence of about 15 to about 50 nucleotide bases present in a nucleic acid of SEQ ID NO: 5.

4. A method for diagnosing systemic lupus erythematosus (SLE) in a human which comprises the steps of:

(a) admixing antibodies from a human to be diagnosed for SLE with a promyelocyte Ehrlichiae Ep group antigen to form an antigen/antibody admixture;

(b) maintaining the antigen/antibody admixture for a time period sufficient for immunoreaction and formation of an immunoreaction product ; and

(c) determining the presence or absence of an immunoreaction product, the presence of an immunoreaction product indicating a diagnosis of SLE.

5. The method according to claim 4, wherein said Ehrlichiae Ep group antigen is from

Ehrlichia equi.

6. The method according to claim 4, wherein said Ehrlichiae Ep group antigen is present in HL60 or KG-I cells.

7. The method according to claim 4 , wherein the presence or absence of said immunoreaction product is detected by an assay method selected from the group consisting of latex agglutination, indirect immunofluorescence, ELISA, solid phase enzyme immunoassay, indirect immunoenzymatic assay, complement fixation, and immunoblot .