Evaluation of the Recombinant Protein TpF1 of Treponema pallidum for Serodiagnosis of Syphilis

Syphilis is a chronic infection caused by Treponema pallidum subsp. pallidum, and diagnosis with sensitive and specific methods is a challenging process that is important for its prevention and treatment. In the present study, we established a recombinant protein TpF1-based indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a Western blot assay for human and rabbit sera. The 20-kDa recombinant protein TpF1 was detected by Western blotting performed with sera from rabbits immunized with recombinant TpF1 and infected with the T. pallidum Nichols strain and T. pallidum clinical isolates but was not detected by Western blotting with sera from uninfected rabbits. The sensitivity of the recombinant protein was determined by screening sera from individuals with primary, secondary, latent, and congenital syphilis (n = 82). The specificity of the recombinant protein was determined by screening sera from uninfected controls (n = 30) and individuals with potentially cross-reactive infections, including Lyme disease (n = 30) and leptospirosis (n = 5). The sensitivities of TpF1-based ELISAs were 93.3%, 100%, 100%, and 100% for primary, secondary, latent, and congenital syphilis, respectively, and the specificities were all 100% for sera from uninfected controls and individuals with potentially cross-reactive infections. In Western blot assays, the sensitivities and specificities of TpF1 for human sera were all 100%. The reactivities of TpF1 with syphilitic sera were proportional to the titers of the T. pallidum particle agglutination (TPPA) assay. These data indicate that the recombinant protein TpF1 is a highly immunogenic protein in human and rabbit infections and a promising marker for the screening of syphilis.     

Lymphocyte Transformation in Syphilis: an In Vitro Correlate of Immune Suppression In Vivo?

Suppression of cellular immunity during primary and secondary infection may explain, in part, the unusual clinical evolution of syphilis. We have previously shown that lymphocytes from normal subjects undergo blastic transformation when exposed in vitro to Treponema refringens. This response was suppressed in patients with syphilis. the suppression being unrelated to serum factors. In the present paper we studied lymphocyte response in vitro to T. refringens, T. reiter, and T. pallidum as well as to monilia and trychophytins. The response to these antigens was suppressed in patients with syphilis although the response to phytohemagglutinin. pokeweed mitogen, and streptolysin was normal. These data support the hypothesis that human infection with T. pallidum is followed by a complex interaction between cellular and humoral immunity, the former being suppressed in primary and secondary stages.     

Activation of macrophages by products of lymphocytes from normal and syphilitic rabbits.

The production of soluble macrophage-activating factors by lymphocytes from syphilitic and normal rabbits was examined. Culture supernatants of splenic lymphocytes cultured with Treponema pallidum antigens or concanavalin A were incubated with rabbit peritoneal macrophages in vitro. The macrophage monolayers were then washed and infected with log-phase Listeria monocytogenes. Activation of the macrophages by lymphocyte products was measured by the ability of the macrophages to resist intracellular multiplication of Listeria and thus survive infection. Macrophages incubated with supernatants of unstimulated lymphocytes or T. pallidum-stimulated lymphocytes from normal rabbits were unable to resist intracellular multiplication of Listeria. Specifically stimulated lymphocytes from syphilitic rabbits and mitogen-stimulated lymphocytes from both normal and syphilitic rabbits demonstrated a clear ability to produce soluble factors which conferred upon macrophages the ability to limit the intracellular growth of the bacteria. Antigen or mitogen alone was unable to activate the macrophages; the presence of lymphocyte products was required.     

Detection of immunoglobulin M antibodies toTreponema pallidum in a modified enzyme-linked immunosorbent assay

The indirect enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin M (IgM) antibodies toTreponema pallidum in sera of syphilitic patients is complicated by false positive reactions due to the interference of IgM rheumatoid factor (IgM-RF) activity and the presence of treponemal IgG antibodies. Another source of error producing false negative results is the competition between treponemal IgG and IgM antibodies for the binding sites on the antigen. To avoid these complications in the indirectTreponema pallidum-specific IgM-ELISA, total IgG was immunoprecipitated from sera of syphilitic patients prior to the assay. The IgM-RF from non-precipitated sera reacted in an IgM-RF-ELISA and in theTreponema pallidum-IgM-specific ELISA with identical titers. After precipitation of total IgG no reactíon of the IgM-RF in the assay could be demonstrated. Competition between IgG and IgM antibodies can be prevented almost completely by the precipitation procedure. The sensitivity and specificity of theTreponema pallidum-specific IgM-ELISA after immunoprecipitation of total serum IgG were shown to be higher than 97 percent.     

Examination of Peripheral Blood Mononuclear Cells and Sera from Thai Adults Naturally Infected with Malaria in Assays of Blastogenic Responsiveness to Mitogenic Lectins and Allogeneic Cell Surface Antigens

We have previously observed that Thai adults who are infected with malaria have a loss of peripheral blood T cells, and that patient sera contain lymphocytotoxic antibodies. In the present study, we examined peripheral blood mononuclear cells from Thai adults naturally infected with Plasmodium falciparum and Plasmodium vivax for the capacity to undergo blastogenesis in response to phytohemagglutinin, concanavalin A, pokeweed mitogen, and allogeneic cell surface antigens in a one-way mixed leukocyte reaction. In addition, sera from actively infected patients were examined with regard to suppressive capabilities toward normal lymphocyte blastogenesis by using the same assays. We found that patient mononuclear cells exhibited normal reactivity to phytohemagglutinin, concanavalin A, and pokeweed mitogen when compared with controls. However, peripheral blood mononuclear cells from patients had a decreased stimulatory capacity in the allogeneic mixed leukocyte reaction, and P. vivax, but not P. falciparum, lymphocytes exhibited decreased responsiveness in the mixed leukocyte reaction. Furthermore, sera from patients with active malaria induced decreased responsiveness by normal mononuclear cells to phytohemagglutinin and concanavalin A, but not pokeweed mitogen; pooled P. falciparum sera caused decreased responsiveness to allogeneic cell surface antigens in the mixed leukocyte reaction. These studies indicate that despite the lost of circulating T cells during the course of infection with malaria, blastogenic responsiveness remains intact, and that sera from patients with malaria are capable of exerting negative immunoregulatory effects.     

Latent syphilis: immunoglobulins reactive in immunofluorescence and other serological tests

Study of sera from 69 patients with untreated or inadequately treated latent syphilis revealed that immunoglobulin (Ig)G antibodies made up the bulk of the fluorescent treponemal antibody-absorption (FTA-ABS)-test reactivity found in the sera. IgM and IgA antibodies also contributed in some cases. Venereal Disease Research Laboratory slide-test reactivity was found in both 19 and 7S serum fractions, whereas Treponema pallidum immobilization-test reactivity was found mainly in the 7S fraction.     

Immunoglobulin Class of Natural Human Antibodies Reactive with Treponema pallidum

To aid the development of serological tests for incubating syphilis, a characterization was made of the background of natural anti-Treponema pallidum antibodies against which the initial immune response to syphilis takes place. Sera from presumed normal persons were studied with monospecific antisera in an indirect fluorescent-antibody procedure. Of 36 sera tested at a 1:5 dilution, all showed IgG reactivity with T. pallidum (Nichols strain), 58% showed IgM reactivity, and 20% showed IgA reactivity. The titer of IgG reactivity was considerably higher than that of the other two immunoglobulins. Heating the sera for 1 hr at 65 C abolished IgA and IgM anti-T. pallidum reactivity, but one-third of the sera retained IgG reactivity. Human Cohn fractions II and III₁ from three commercial sources contained mostly IgG antibodies reactive with T. pallidum, but IgM and IgA antibodies were also present. IgG reactivity was found in a pool of presumably normal maternal sera from 20 mothers and in a corresponding pool of umbilical cord serum from their infants. IgM and IgA reactivities were present only in the maternal serum pool.     

Characterization and Serologic Analysis of the Treponema pallidum Proteome

Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease characterized by widespread tissue dissemination and chronic infection. In this study, we analyzed the proteome of T. pallidum by the isoelectric focusing (IEF) and nonequilibrating pH gel electrophoresis (NEPHGE) forms of two-dimensional gel electrophoresis (2DGE), coupled with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis. We determined the identity of 148 T. pallidum protein spots, representing 88 T. pallidum polypeptides; 63 of these polypeptides had not been identified previously at the protein level. To examine which of these proteins are important in the antibody response to syphilis, we performed immunoblot analysis using infected rabbit sera or human sera from patients at different stages of syphilis infection. Twenty-nine previously described antigens (predominantly lipoproteins) were detected, as were a number of previously unidentified antigens. The reactivity patterns obtained with sera from infected rabbits and humans were similar; these patterns included a subset of antigens reactive with all serum samples tested, including CfpA, MglB-2, TmpA, TmpB, flagellins, and the 47-kDa, 17-kDa, and 15-kDa lipoproteins. A unique group of antigens specifically reactive with infected human serum was also identified and included the previously described antigen TpF1 and the hypothetical proteins TP0584, TP0608, and TP0965. This combined proteomic and serologic analysis further delineates the antigens potentially useful as vaccine candidates or diagnostic markers and may provide insight into the host-pathogen interactions that occur during T. pallidum infection.Syphilis is a multistage progressive disease caused by the spirochete Treponema pallidum subsp. pallidum and is characterized by localized, disseminated, and chronic stages. Manifestations include the development of a localized lesion called a chancre during the primary stage and disseminated skin lesions and meningovascular syphilis during the secondary stage, followed by a period of latency lasting from months to decades. Chronic, debilitating symptoms develop during the tertiary stage, including granuloma-like lesions called gummas, neurosyphilis, and cardiovascular syphilis (38). Although syphilis can be successfully treated by antibiotics, it remains a significant public health problem, with an estimated 12 million new cases per year worldwide (41).Continued improvement of diagnostic tests (particularly point-of-care tests) as well as the development of an effective vaccine for syphilis would aid greatly in the control of syphilis (4, 6). T. pallidum research, including the identification of antigens, has been hindered by the inability to culture the bacterium continuously in vitro, necessitating the propagation of organisms by experimental rabbit infection (28). In addition, the fragility and low protein content of the T. pallidum outer membrane have complicated the identification of surface proteins potentially useful in vaccines (5, 28).The T. pallidum genome sequence (15) provides an additional tool for the analysis of potential antigens. The 1.14-Mb T. pallidum chromosome contains 1,039 open reading frames (ORFs) encoding predicted protein products, a smaller number than for any other spirochete genome sequenced to date (15). The average size of predicted proteins is 37,771 Da, ranging from 3,235 to 172,869 Da. Analysis of the translated genome of T. pallidum predicts an unusually basic proteome, with a mean pI of 8.1 and median pI of 8.5, with 66% of proteins having pIs of >7.0 (23). Small genome size and a predominance of basic proteins are more common in parasitic microorganisms, and the latter is thought to facilitate interaction of the organism with its host (20). Other pathogenic spirochetes also tend to have basic proteins; for example, the proteome of Borrelia burgdorferi has a mean pI of 8.36 and median pI of 9.03 (14, 29a), and 69% of Leptospira interrogans serovar Lai strain 56601 proteins have pIs greater than 7.0 (24, 33). A recent analysis of the T. pallidum genome indicates the presence of 46 putative lipoproteins, many fewer than the 127 predicted for B. burgdorferi (34).The availability of the genome sequence made it possible to examine predicted T. pallidum ORFs for potential suitability as diagnostic or immunization tools. McKevitt et al. (22) and Brinkman et al. (3) created a protein expression library of 900 of the 1,039 T. pallidum proteins predicted from the genome sequence and examined the serologic reactivity of these proteins by enzyme-linked immunosorbent assays (ELISAs). They identified 106 antigens reactive with rabbit sera and 34 antigens reactive with sera from syphilis patients. This set of antigens was termed the T. pallidum immunoproteome. This approach permits identification of low-abundance T. pallidum antigens, since they may be expressed as recombinant proteins in much larger quantities. Conversely, proteins that are poorly expressed in Escherichia coli or do not fold correctly may not be detected, leading to false-negative results.To provide a complementary set of data regarding the T. pallidum immunoproteome, we have performed proteomic analysis of T. pallidum proteins expressed during experimental rabbit infection. We used isoelectric focusing (IEF) and nonequilibrating pH gel electrophoresis (NEPHGE) forms of two-dimensional gel electrophoresis (2DGE) coupled with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis to identify T. pallidum polypeptides. Immunoblotting was subsequently used to identify antigens reactive with infected rabbit sera (IRS) and with human sera obtained at different stages of syphilis. This approach may permit identification of antigens that are not expressed well in E. coli and provides a more accurate picture of the level of protein expression in the intact organism. We have thereby characterized most of the major T. pallidum proteins expressed in infected tissue and identified a set of antigens reactive at all stages of infection, which could potentially be useful for the development of improved immunodiagnostic tests or for vaccines.     

Molecular analysis of immunoglobulins M and G immune response to protein antigens of Treponema pallidum in human syphilis.

Protein antigens of Treponema pallidum precipitated by immunoglobulin M (IgM) and IgG antibodies of sera from patients with untreated primary and secondary syphilis as well as treated secondary syphilis were characterized on a molecular basis. T. pallidum was labeled internally with [35S]methionine and solubilized in 0.1% sodium dodecyl sulfate-1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12.5% gels followed by autoradiography revealed 32 distinct proteins with molecular weights between 13,500 and 200,000. Twenty-three proteins of T. pallidum with molecular weights between 15,500 and 115,000 were identified as antigens by double antibody radioimmunoprecipitation with IgM and IgG antibodies of sera from syphilitic patients. The molecular analysis of the IgM and IgG immune response to T. pallidum in human syphilis is in accord with earlier immunological observations. Finally, utilizing syphilitic human sera, we characterized 15 protein antigens of T. pallidum that are common to Treponema phagedenis by partial absorption of IgM and IgG antibodies with an ultrasonicate of T. phagedenis.     

SERUM IMMUNOGLOBULINS AND LYMPHOCYTE SUBPOPULATIONS DERANGEMENT IN TURNER'S SYNDROME

Abnormalities of the proportions of peripheral blood lymphocyte subpopulations and of immunoglobulin serum levels were found in twenty patients affected by Turner's syndrome. A slight but significantly decreased percentage of circulating T and B cells, an increased percentage of null cells and a decreased in vitro, responsiveness of lymphocytes to phytohaemagglutinin, concanavalin A and pokeweed mitogen were found in Turner's syndrome patients. IgG serum level was found significantly decreased in comparison with age-matched fifty-seven normal males and fifty-seven normal females and IgM serum level was intermediate between female and male values; Turner's syndrome patients with monosomy had an IgM serum concentration very close to male values. The derangement of T and B lymphocyte subpopulations, probably related to the aneuploidy, does not seem to be a severe one but it could account for the immunoglobulin abnormalities and for the association of Turner's syndrome with immunological disorders such as autoimmune diseases. The role of X chromosome on IgM serum level is discussed.     

Molecular Characterization of Treponema pallidum mcp2, a Putative Chemotaxis Protein Gene

The nucleotide sequence of the Treponema pallidum mcp2 gene was determined. mcp2 encodes a 45.8-kDa protein whose deduced amino acid sequence has significant homology with the C-terminal region of bacterial methyl-accepting chemotaxis proteins (MCPs). The Mcp2 N terminus lacks the hydrophobic transmembrane regions present in most MCPs. An Mcp2 fusion protein was strongly reactive with antibody (HC23) to the highly conserved domain of MCPs and with rabbit syphilitic serum. Antibody HC23 reacted with six T. pallidum proteins, including a 45-kDa protein that may correspond to Mcp2. This protein was present in the aqueous phase from T. pallidum cells that were solubilized with Triton X-114 and phase partitioned.     

Western Immunoblotting with Five Treponema pallidum Recombinant Antigens for Serologic Diagnosis of Syphilis

Five immunodominant Treponema pallidum recombinant polypeptides (rTpN47, rTmpA, rTpN37, rTpN17, and rTpN15) were blotted onto strips, and 450 sera (200 from blood donors, 200 from syphilis patients, and 50 potentially cross-reactive) were tested to evaluate the diagnostic performance of recombinant Western blotting (recWB) in comparison with in-house whole-cell lysate antigen-based immunoblotting (wclWB) and T. pallidum hemagglutination (MHA-TP) for the laboratory diagnosis of syphilis. None of the serum specimens from blood donors or from potential cross-reactors gave a positive result when evaluated by recWB, wclWB, or MHA-TP. The evaluation of the immunoglobulin G immune response by recWB in sera from patients with different stages of syphilis showed that rTmpA was the most frequently identified antigen (95%), whereas only 41% of the specimens were reactive to rTpN37. The remaining recombinant polypeptides were recognized as follows: rTpN47, 92.5%; rTpN17, 89.5%; and rTpN15, 67.5%. The agreement between recWB and MHA-TP was 95.0% (100% with sera from patients with latent and late disease), and the concordance between wclWB and MHA-TP was 92.0%. The overall concordance between recWB and wclWB was 97.5% (100% with sera from patients with secondary and late syphilis and 94.6 and 98.6% with sera from patients with primary and latent syphilis, respectively). The overall sensitivity of recWB was 98.8% and the specificity was 97.1% with MHA-TP as the reference method. These values for sensitivity and specificity were slightly superior to those calculated for wclWB (sensitivity, 97.1%, and specificity, 96.1%). With wclWB as the standard test, the sensitivity and specificity of recWB were 98.9 and 99.3%, respectively. These findings suggest that the five recombinant polypeptides used in this study could be used as substitutes for the whole-cell lysate T. pallidum antigens and that this newly developed recWB test is a good, easy-to-use confirmatory method for the detection of syphilis antibodies in serum.     

Characterization of the Glycoprotein Antigens Which Mediate the Schistosoma japonicum Circumoval Precipitin Reaction

The circumoval precipitin test is a serological test used for diagnosis of schistosomiasis japonica. Soluble egg antigens of Schistosoma japonicum block the formation of the circumoval precipitin by serum from infected humans. Consequently, circumoval precipitin inhibition was used to monitor purification of the soluble egg antigens of S. japonicum. Crude egg antigens were separated into protein and glycoprotein fractions by lectin chromatography on concanavalin A Sepharose. The glycoprotein fraction produced two intense precipitin lines upon immunodiffusion analysis with human chronic infection sera. The protein fraction produced two faint precipitin lines which did not cross-react with those of the glycoprotein fraction. The glycoprotein fraction contained 90% of the circumoval precipitin inhibitory activity. Isoelectric focusing of ¹²⁵I-labeled concanavalin A Sepharose fractions revealed at least four groups of potential S. japonicum antigens, termed JAG I, II, and III, and a JAG IV complex. These had isoelectric points ranging from 3.2 to 6.7. In these respects, the S. japonicum egg antigen glycoproteins are similar to those of Schistosoma mansoni. The glycoproteins were further separated by diethylaminoethyl ion-exchange chromatography. On immunodiffusion analysis it was found that one of the strong Ouchterlony precipitin lines was associated with glycoproteins that did not adsorb to diethyl-aminoethyl columns, whereas the second Ouchterlony precipitin was heterodisperse, being present in the first, second, and third of the four peaks eluted from the diethylaminoethyl column. Immunoelectrophoresis of the diethylaminoethyl fractions demonstrated that the antigen present in highest concentration in soluble egg antigen glycoproteins, JAG II, was extremely heterodisperse in its behavior on diethylaminoethyl columns. This is unlike the S. mansoni antigens which can be easily separated by diethylaminoethyl ion-exchange chromatography.     

Elevation of CD8+ CD11b+ Leu-8− T cells is associated with the humoral immunodeficiency in myeloma patients

Recurrent bacterial infections due to humoral immunodeficiency are an important cause of death in myeloma patients. Recent data indicate that CD8⁺ T lymphocytes and a reduction of T helper type 1 cells with disease progression may be involved in the regulation of polyclonal immunoglobulin secretion. In mixed lymphocyte cultures derived from peripheral blood mononuclear cells (PBMC) of 24 myeloma patients with reduced immunoglobulin serum levels we investigated the association of CD4⁺ and CD8⁺ T cell subsets and immunoglobulin-secreting B cells (ISC) upon mitogenic stimulation with pokeweed mitogen (PWM) and concanavalin A (Con A). In supernatants of cultured PBMC of myeloma patients the spontaneous secretion of the type 1 cytokine interferon-gamma was reduced. After PWM stimulation reduced numbers of polyclonal ISC were found in 79% of patients, and monoclonal ISC were observed in 12% of patients. After Con A stimulation, again formation of polyclonal ISC was reduced, but monoclonal ISC were found in 41% of patients. Elevation of monoclonal and reduction of polyclonal ISC after stimulation with Con A were associated with an increase of CD8⁺ CD11b⁺ Leu-8⁻ T cells (P < 0.05). We conclude that the elevated numbers of CD8⁺ CD11b⁺ Leu-8⁻ T cells play a role in the stimulation of monoclonal and suppression of polyclonal immunoglobulin secretion in myeloma patients.     

The role of cell-mediated immune mechanisms in syphilis in Ethiopia

The lymphocyte transformation test was used to assess cell-mediated immune reactivity in 107 Ethiopian patients with syphilis. Lymphocytes from patients with early syphilis were unreactive to the Nichols strain of Treponema pallidum, which was in marked contrast to previous findings in similar patients in England. Lymphocytes obtained from patients with late syphilis, however, were reactive. The responses elicited by a strain of T. pallidum isolated from an Ethiopian with early syphilis did not differ from those with the usual Nichols strain. About half the patients with early syphilis who had received antibiotic treatment for 8 days showed an increase of lymphocyte reactivity towards T. pallidum, although responses to PPD and PHA were unchanged.

Plasma from patients with syphilis was examined for its capacity to inhibit lymphocyte responses in vitro. Although plasma from people with late (cardiovascular) syphilis did not differ from controls, plasma from patients with early syphilis inhibited the responses of their own cells to both PPD and PHA. The inhibitory effect on PHA responses was abrogated after the patients had received antibiotic treatment for 1 week.

The significance of the differences in lymphocyte reactivity observed between Ethiopians with syphilis and their counterparts in England is discussed with regard to a possible explanation of the differences in the natural history of the disease in the two countries.

     

The absence of differences in cellular immunity in elderly individuals with and without delayed local cutaneous reactions to influenza vaccine.

Two groups of elderly subjects who received bivalent influenza virus vaccine were identified. Those in Group I manifested marked local reactions, suggestive of delayed hypersensitivity reactions, whereas those in Group II, matched with Group I for vaccine preparation, had no cutaneous reactions. In vitro assays of immune function were performed on pairs of subjects from the two groups and included serum immunoglobulin and complement levels, antibody response to vaccine, lymphocyte transformation to mitogens (phytohemagglutinin, pokeweed mitogen and concanavalin A) and antigens (streptokinase-streptodornase, influenza virus vaccine antigens and multiple mixed lymphocytes), and polymorphonuclear leukocyte chemotactic responsiveness. There was no demonstrable correlation between delayed local cutaneous reaction and preservation of in vitro cell-mediated immune function in these elderly individuals.     

Enhancement of in vitro lymphocyte response by neuraminidase

In vitro blastogenic response of sensitized human lymphocytes to specific antigens was significantly enhanced by Vibrio cholerae neuraminidase treatment. No such enhancement was noted when the unsensitized lymphocytes were treated with this enzyme. An enhancement effect of neuraminidase on the lymphocyte response to concanavalin A or pokeweed mitogen (weak mitogens) was also noted; the enzyme had no effect on phytohaemagglutinin (strong mitogen)-induced blasto-genesis. The increased reactivity of lymphocytes is probably related to changed physical and functional properties of the cells after treatment with neuraminidase.     

Identification and preliminary characterization of Treponema pallidum protein antigens expressed in Escherichia coli.

We have previously described the construction in Escherichia coli K-12 of a hybrid plasmid colony bank of Treponema pallidum (Nichols strain) genomic DNA. By screening a portion of this bank with an in situ immunoassay, we identified six E. coli clones that express T. pallidum antigens. In this study, the recombinant plasmids from each of these clones have been analyzed in E. coli maxicells and have been found to encode a number of proteins that are not of vector pBR322 origin and are, therefore, of treponemal origin. In each case, several of these proteins can be specifically precipitated from solubilized maxicell extracts by high-titer experimental rabbit syphilitic serum. Certain of these proteins are also precipitated by high-titer latent human syphilitic sera (HSS). The T. pallidum DNA inserts in these plasmids range in size from 6.2 to 14 kilobase pairs, and from the restriction patterns of the inserts and the protein profiles generated by each plasmid in maxicells, it is apparent that we have recovered a total of four unique clones from our colony bank. Recombinant plasmids pLVS3 and pLVS5 were of particular interest. Plasmid pLVS3 encodes three major protein antigens with molecular weights of 39,000, 35,000, and 25,000. These three proteins, which were not recognized by pooled normal human sera, were efficiently precipitated by most secondary HSS, latent HSS, and late HSS tested. These proteins were also precipitated, although somewhat inefficiently, by most primary HSS tested. Plasmid pLVS5 encodes a major protein antigen with a molecular weight of 32,000 and several minor protein antigens that, although efficiently precipitated by experimental rabbit syphilitic serum, were generally not recognized by the various HSS tested. Evidence is presented indicating that the protein antigens encoded by plasmids pLVS3 and pLVS5 are specific for pathogenic treponemal species. We have also demonstrated that immunoglobulin G antibodies directed against these protein antigens can be detected in rabbits experimentally infected with T. pallidum Nichols as early as 11 days postinfection.     

Human milk contains proteins that stimulate and suppress T lymphocyte proliferation.

The modulatory effect of human milk proteins from colostrum and late milk on the proliferative response of human T lymphocytes activated by mitogens (OKT3 and leucoagglutinin from Phaseolus vulgaris) and alloantigens was studied. High concentrations (10-100 micrograms/ml) of crude colostral milk proteins had an inhibitory effect on T cell growth while low concentrations (0.1-1 microgram/ml) enhanced T cells growth. In contrast, proteins from late milk did not inhibit T lymphocyte proliferation while the enhancing effect was retained. Colostrum was fractionated by ammonium sulphate precipitation and gel filtration on sepharose 6B. The inhibitory activity was recovered in a protein fraction containing lactoferrin as its major component. Lactoferrin was, however, not responsible for the observed inhibition. On the contrary, lactoferrin in most cases augmented the proliferative response induced by polyclonal activators. The inhibitory activity was found to bind concanavalin A-sepharose suggesting an association with glycoprotein. Inhibitory fractions contained glycoproteins of the following molecular sizes 26, 74/76 (doublet), 84, 145 and 160 kD under reducing conditions. The inhibitory effect appeared to be lymphocyte specific since the active fraction did not inhibit the growth of tissue culture cells (HeLa cells and human fibroblasts) or bacteria. Furthermore, the fraction was not toxic for lymphocytes. The inhibitory colostrum factor may prevent the newborn from overreacting immunologically against the environmental antigens encountered at birth.     

Alterations in the Distribution and Proliferative Responses of Rhesus Monkey Peripheral Blood and Spleen Cells During Malaria (Plasmodium knowlesi) Infection

Rhesus monkeys are used frequently as animal models in malaria research, but few studies have evaluated lymphocyte functions in these animals after experimental infections with the primate malarial parasite Plasmodium knowlesi. In this study, the distribution and mitogen responses of mononuclear cells in the peripheral blood and spleens of 16 P. knowlesi-infected rhesus monkeys were followed. All animals included in the study developed acute infections and were bled out with parasitemias of more than 50%. With progression of the infection, alterations in the peripheral blood mononuclear cells were observed, including decreases in the percentage of T cells (measured by E rosette formation) and the total numbers of E and EAC rosette-forming cells per cubic millimeter. In addition, peripheral blood mononuclear cells displayed reduced responses to mitogen stimulation with phytohemagglutinin, concanavalin A, and pokeweed mitogen. Peripheral blood mononuclear cells from infected animals showed similar reductions in mitogen responses when cultured in media containing 15% autologous pre- or postinfection plasma. The mitogen responses of spleen cells did not appear to be affected, but a significant reduction in the proportion of splenic T cells was observed. These lymphocyte changes in P. knowlesi-infected rhesus monkeys are similar to those reported for mice with acute rodent malaria and for humans with chronic Plasmodium falciparum infections.