Gail S. Habicht

Spirochetes Isolated from the Blood of Two Patients with Lyme Disease

We isolated spirochetes from the blood of 2 of 36 patients in Long Island and Westchester County, New York, who had signs and symptoms suggestive of Lyme disease. The spirochetes were morphologically similar and serologically identical to organisms recently found to infect lxodes dammini ticks, which are endemic to the area and have been epidemiologically implicated as vectors of Lyme disease. In both patients, there was a rise in specific antispirochetal antibodies in paired specimens of serum. We conclude that the l. dammini spirochete has an etiologic role in Lyme disease.

Babesiosis in splenectomized adults: Review of 22 reported cases

Since 1957, there have been 22 reported cases of human babesiosis in splenectomized persons, representing about one third of all clinical human babesiosis. Splenectomy had been performed one month to 36 years (mean 8.7 years, median 6.0 years) earlier for a variety of reasons. Four of the seven European cases were from Babesia divergens whereas 12 of the 15 United States cases were from B. microti. Most of the 22 patients had moderate to severe clinical disease including hemolytic anemia, yet all but six recovered. Three patients had transfusion-acquired babesiosis. Treatments employed included the use of chloroquine, quinine, pyrimethamine, pentamidine, clindamycin, dialysis, and exchange transfusion. Splenectomized and/or otherwise immunocompromised hosts should be advised to avoid visiting endemic areas for babesiosis such as Nantucket Island or Marthas Vineyard in Massachusetts and Shelter Island and other parts of Long Island, New York. Babesiosis must be considered as one of the not uncommon organisms responsible for the postsplenectomy sepsis syndrome and one for which there is no current prophylaxis.

Characterization of interleukin-1 activity in tunicates☆

1. Eight North American species of tunicates were examined for the presence of interleukin-1 (IL-1) like activity. 2. The tunicates studied produce molecules with readily detectable lymphocyte activation factor (LAF) activity. 3. G50 column chromatography separated molecular species that were directly mitogenic for thymocytes (mol. wt greater than 50,000) from those that were comitogenic in an IL-1 assay (mol. wt 20,000). 4. Tunicate fractions with LAF activity induced increased vascular permeability in rabbit skin. 5. Tunicate LAF activity was neutralized by polyclonal anti-human IL-1 antisera. 6. These data further support the conclusion that IL-1 is an ancient and functionally conserved molecule.

Primitive cytokines: harbingers of vertebrate defense

The evolution of the immune system has awarded cytokines a key role as coordinators of the immune response. The exquisite action of cytokines in fine tuning and controlling the response raises the question of whether or not these molecules have been highly conserved through evolution. Gregory Beck and Gail Habicht have isolated and characterized two major cytokines, interleukin 1 and tumor necrosis factor, from invertebrates. In this article, they speculate on the possible function of these molecules and on the existence of other cytokines in invertebrates.

Pathogenesis of Lyme neuroborreliosis in the rhesus monkey: the early disseminated and chronic phases of disease in the peripheral nervous system.

The histopathologic and immunohistochemical features of early and late neuroborreliosis of the peripheral nervous system were investigated in rhesus macaques infected with the JD1 strain of Borrelia burgdorferi. Infection was proven by culture or polymerase chain reaction analysis of skin biopsies and indirectly by Western blot analysis. Three months after infection, neuritis involving multiple nerves was the most consistent neurologic manifestation. Both macrophages and B lymphocytes but not T lymphocytes were present in the cellular infiltrates. Axonal structures surrounding infiltrates had changes consisting of demyelination and axonal phagocytosis. Some of the Schwann cells in lesions stained with anti-nitrotyrosine and anti-tumor necrosis factor-alpha antibodies. B. burgdorferi, or antigens thereof, were visualized immunohistochemically within macrophages. Forty-six months after infection, the most common changes were regenerative, whereas neuritis was infrequent. Aberrant axonal regeneration, irregularly sized myelinated fibers, and fibrosis were frequently observed. Possible mechanisms to explain the appearance and subsidence of Lyme neuritis are discussed.

Isolation, preliminary chemical characterization, and biological activity of Borrelia burgdorferi peptidoglycan☆

Peptidoglycan (PG), an essential cell wall polymer of most bacteria, has been isolated from many species of spirochetes. Our interest in the host response to Borrelia burgdorferi led us to isolate and characterize its PG. Extracted cells were solubilized with warm 1% SDS followed by digestion with proteases. Amino acid analysis of the isolated PG demonstrated the presence of alanine, glycine, glutamic acid, and ornithine as occurs in other spirochetes and bacteria. Intense erythematous reactions were observed after id injection of 10 micrograms of PG into normal human skin. PG was not mitogenic for human peripheral blood mononuclear cells. Murine splenocytes of certain strains responded to the PG, but only at concentrations of 25 micrograms/ml or more. PG stimulated macrophages to produce interleukin 1. Sixteen micrograms of PG injected iv into rabbits produced biphasic fevers. These observations on the in vitro and in vivo activities associated with the cellular components of the B. burgdorferi spirochete give further insight to how a small number of invading organisms can cause a multisystemic disease such as Lyme disease.

Nitric oxide production by coelomocytes of Asterias forbesi.

Vertebrate mononuclear phagocytes produce a plethora of molecules involved in host defense. Among the most potent are the reactive oxygen and nitrogen intermediates. Coelomocytes from invertebrates subserve many of the same functions. In order to determine whether invertebrate phagocytes employ reactive nitrogen intermediates, we investigated the effect of various nonspecific stimulators and invertebrate interleukin (IL)-1alpha- and beta-like molecules on nitric oxide (NO) production. Elevated NO release by stimulated coelomocytes was seen after 24 h. Incubation of stimulated coelomocytes in the presence of arginine analogs inhibited NO release. When invertebrate IL-1-like molecules were added to the coelomocytes, they stimulated the release of NO. Western blot analysis using a polyclonal rabbit antiserum to murine NO synthase detected a band at approximately 125 kDa. These data indicate that coelomocytes are capable of producing and releasing NO and that NO is a chemical mediator that has been conserved as a host defense weapon of phagocytes through evolutionary time.

A role for interleukin-1 in the pathogenesis of Lyme disease.

Summary Interleukin-1 (IL-1) is the major immunoregulatory molecule produced by macrophages in response to a variety of environmental insults including chemicals, phagocytosis, bacteria, and bacterial products. Macrophages stimulated by Borrelia burgdorferi produced large quantities of IL-1 when spirochetes were added to macrophages at a ratio of 10 spirochetes per macrophage. The release of IL-1 was dose dependent: a single spirochete per macrophage was sufficient to produce significant quantities of IL-1. Spirochetal lipopolysaccharide was not required for this activity in that polymyxin B in the spirochete-macrophage culture had no effect on IL-1 production. Normal murine fibroblasts cultured with this IL-1 were shown to have an increased rate of DNA synthesis and an increase in secreted collagenase. IL-1 was found in joint fluids from Lyme disease patients. When IL-1 was injected intradermally into the backs of rabbits, the injection sites became indurated, erythematous, and warm to the touch after 4 hrs and annular lesions much like those of erythema chronicum migrans were seen in some animals after 24 hrs. B. burgdorferi is a powerful inducer for IL-1 in vitro , and it is reasonable to presume that it acts similarly in Lyme disease patients. Our results suggest that IL-1 in turn, may play a role in many of the clinical manifestations of Lyme disease.

Isolation of the outer envelope from Borrelia burgdorferi

Borrelia burgdorferi consists of an inner protoplasmic cylinder, containing the genome and cytoplasmic elements, surrounded by a number of axial filaments, all completely encased within a multiple-layered outer envelope structure (OE). In this study, a sodium dodecyl sulfate-mediated technique was used to isolate the OE from Borrelia burgdorferi in an attempt to better understand this structure in terms of its antigenic reactivity to Lyme disease patient sera. Electron microscopic evidence suggested that the OE product was relatively free of other spirochete cellular components. SDS-PAGE analysis indicated that the electrophoretic pattern of the OE was consistent with that of the remaining protoplasmic cylinder (PC) and whole spirochete controls. Antigenic determinants in the OE were recognized by sera from Lyme disease patients in Western blots. Chemical analysis of the OE revealed a composition of 45.90% protein, 50.75% lipid and 3.33% carbohydrate. The OE comprised 16.5% by lyophilized dry weight of the whole spirochete. Antigenic determinants located within or associated with this structure are likely to play a significant role in the development of immunity in the infected host.

Preparation of conjugated erythrocytes for long term use in hemolytic plaque assays, complement fixation studies, or passive hemagglutinations: A comparative study of several methods

A variety of conjugation procedures, with and without glutaraldehyde stabilized erythrocytes, was employed to couple human IgG to sheep erythrocytes. The efficiency of conjugation, the recovery of cells, and the extent of labeling are noted. These conjugated erythrocytes were then tested for suitability in the hemolytic plaque assay, in a passive hemagglutination test, in a complement assay, and in the detection of rheumatoid factor. Although several methods were useful in a variety of situations, it was found that conjugation using a water soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) at low temperature (4 degrees C) produced a uniformly acceptable product that was stable and reproducible over a seven week period. The use of this method for a diversity of antigens or haptens is discussed.