[Sweet's syndrome induced by pegfilgrastim during a myelodysplastic syndrome AREB2: A case report]. / Syndrome de Sweet induit par le pegfilgrastim au cours d'un syndrome myélodysplasique AREB2 : à propos d'un cas clinique.

INTRODUCTION: Sweet's syndrome is an acute neutrophilic dermatosis characterized by abrupt onset of skin lesions accompanied by fever, arthralgia, leukocytosis and diffuse neutrophilic infiltration of the dermis, as well as an excellent response to corticosteroid therapy. CASE REPORT: A 46-year-old patient with myelodysplastic syndrome was admitted for chemotherapy. On the eighth day of chemotherapy, he received a single dose of pegfilgrastim. Three days later, he developed pyrexia, conjunctivitis, arthralgia and erythematous and painful papulo-nodular lesions. Broad-spectrum empiric antibiotic therapy was started but the patient's condition deteriorated. Biology showed pancytopenia and inflammatory syndrome. Microbiological tests, autoimmune serologies and chest-computed tomography were negative. Cutaneous biopsy was compatible with Sweet's syndrome. A diagnosis of Sweet's syndrome induced by pegfilgrastim was made and intravenous corticosteroid therapy was started with a rapid favorable outcome. CONCLUSION: Sweet's syndrome is a rare adverse effect of G-CSF.

[Ovarian metastasis and lung adenocarcinoma: a case report]. / Métastase ovarienne et adénocarcinome pulmonaire: observation d'un cas clinique.

Ovarian metastasis as first dissemination site of a lung adenocarcinoma has not been described in the literature. We report the case of a 61-year-old woman who had a pneumectomy for a centrally located lung adenocarcinoma, which was discovered on a routine chest X-Ray. During the follow-up, a Positron Emission Tomography (PET)-Scan showed a hypercaptation in the pelvic region. Abdominal CT-scan confirmed the presence of a mass which was compatible with a primary ovarian tumor. The patient underwent a hysterectomy and bilateral salpingo-oophorectomy. Pathology reported an adenocarcinoma. Immunohistochemical staining revealed cells expression for Thyroid Transcription Factor 1 (TTF-1), cytokeratin 7 (CK-7) and focally cytokeratin 20 (CK-20). Clinical course, pathological and immunohistochemical data concluded to the diagnosis of ovarian metastasis of the lung adenocarcinoma. In conclusion, in the differential diagnosis of an ovarian metastasis, clinicians should not forget the lung as primary site since epidemiologic data of lung cancer in women show progressive incidence.

What are the immunologically active components of bacille Calmette-Guérin in therapy of superficial bladder cancer?

The subcomponents of bacille Calmette-Guérin (BCG) involved in the mechanism of action of intravesical BCG immunotherapy used for prophylaxis of superficial bladder cancer recurrences have been poorly investigated. We purified various BCG subcomponents and analyzed in vitro their ability to enhance a Th1 polarized immune response as well as to increase lymphocyte-mediated cytotoxicity against bladder tumors. Human peripheral blood mononuclear cells (PBMCs) from healthy purified protein derivative-positive subjects were incubated for 7 days with whole BCG and various fractions (BCG cell wall, plasma membrane, cytosol, purified polysaccharides as glucan or arabinomannan, purified native proteins from BCG culture filtrate, recombinant 22 kDa protein, phosphate transporter PstS-2 and -3 proteins). IFN-gamma, IL-12, IL-2, and IL-6 production by stimulated PBMCs was compared to unstimulated controls and the phenotype of expanded cells analyzed by flow cytometry (FACS analysis). A (51)Cr-release assay monitored the cytotoxicity of amplified effector cells against T24 bladder tumor cells. Live BCG and most of its subcomponents (with the exception of cytosol, PstS-2 and -3) significantly enhanced IFN-gamma and IL-12 secretion, expanded CD3(-)CD56(+) cells and the non-MHC-restricted cytotoxicity against bladder tumor cells compared to unstimulated controls (all P < 0.001, t-test). IL-2 receptor blockage resulted in a clear reduction in the cytotoxic activity of stimulated PBMCs. Numerous BCG subcomponents thus provide positive stimuli for Th1 cell differentiation and enhance in vitro, non-MHC-restricted cytotoxicity against bladder tumor cells. Our findings provide the basis for the therapeutic use of several of these subfractions in experimental animal models bearing bladder tumors.

What is the optimal regimen for BCG intravesical therapy? Are six weekly instillations necessary?

OBJECTIVE: For more than 20 years, BCG intravesical therapy schedule has included 6 weekly instillations. Very few studies have, however, analyzed the rationale of this regimen. We previously demonstrated that intravesical BCG induced an increased peripheral immune response against mycobacterial antigens as compared to pretreatment values. In the present work, we have studied the weekly evolution of this immune response induced by intravesical BCG instillations. MATERIALS AND METHODS: The evolution of the lymphoproliferative response of peripheral blood mononuclear cells against BCG culture filtrate (CF), tuberculin (PPD) and BCG extract (EXT) was tested before, every week during the BCG instillations and at 3 and 6 months follow-up in 9 patients with superficial bladder cancer treated with 6 weekly BCG instillations. Lymphoproliferation was measured by means of a tritiated thymidine incorporation test. RESULTS: A significant increase in the lymphoproliferative response against PPD, CF and EXT was observed in 9, 8 and 7 of the 9 patients, respectively, as compared to pre-BCG values. The maximal lymphoproliferation was achieved after 4 instillations in 4/5 patients initially reactive against mycobacterial antigens whereas 2 of 4 initially nonreactive patients required 6 instillations. At 6 months' follow-up, lymphoproliferation against BCG and the other mycobacterial antigens returned to pre-BCG values in all patients. In 3 patients who received additional instillations because of tumor recurrence within 1 year of follow-up, the maximum immune response was observed already after 2 instillations. CONCLUSION: In most patients, the maximal peripheral immune response is already observed after 4 weekly instillations. However, patients not previously immunized against mycobacterial antigens may require 6 weekly instillations to achieve a maximum stimulation level. Our data support the need to further evaluate the role of this status before starting BCG instillations. It could be of interest to study whether 6 BCG instillations are really necessary in patients previously immune against mycobacterial antigens.

Correlation and prognostic significance of p53, p21WAF1/CIP1 and Ki-67 expression in patients with superficial bladder tumors treated with bacillus Calmette-Guerin intravesical therapy.

PURPOSE: We determine if, before intravesical bacillus-Calmette Guerin (BCG) therapy, p53, p21WAF-1-CIP1 (a critical downstream effector of p53 pathway of cell growth control, inhibiting cyclin dependent kinases) and the cell proliferation marker Ki-67 (MIB-1) could be used as prognostic markers of response to BCG in patients with superficial bladder tumors. MATERIALS AND METHODS: The study included 47 patients with superficial bladder tumors at high risk for recurrence or progression treated with 6 weekly intravesical BCG instillations. We analyzed p53, p21 and Ki-67 on paraffin embedded samples by immunohistochemistry and the percentage of positive cells was determined in a blinded fashion. Quantitative immunostaining was analyzed in relation to time to recurrence and progression using univariate or multivariate analysis and the Kaplan-Meier method. RESULTS: During a mean followup of 24.6 months 23 of the 47 patients (48.9%) presented with tumor recurrence and 10 (21.2%) had later progression to invasive disease. A p21 over expression (greater than 10%) was observed in 23 tumors (48.9%) and positively correlated with p53 (p = 0.0097) but not with Ki-67 (p = 0.327). Of the tumors 18 (38.2%) were p53 and p21 negative. Among p21 positive tumors 15 (65.2%) were p53 and p21 positive, suggesting that p21 may also be regulated by p53 independent pathways. However, p53 did not act as a predictor of recurrence or progression. In contrast, using Kaplan-Meier curves p21 over expression (greater than 10%) and Ki-67 at a 25% cutoff were associated with shorter recurrence-free survival (both p = 0.02 log rank test) but they did not predict additional information about risk of progression. However, multivariate analysis failed to demonstrate any independent prognostic value for p21 or Ki-67 in contrast to tumor stage. CONCLUSIONS: Our results indicate that p21WAF-1-CIP1 seems to be regulated by p53 independent pathways in superficial bladder cancer. The present study did not indicate an independent prognostic significance in patients treated with BCG for p53, p21WAF-1-CIP1 or Ki-67 markers. Larger prospective studies are needed to evaluate further the independent value of these biological markers in superficial bladder cancer management.

Superficial bladder tumors and increased reactivity against mycobacterial antigens before bacillus Calmette-Guerin therapy.

PURPOSE: The precise mechanism of action of bacillus Calmette-Guerin (BCG) in bladder cancer treatment remains poorly understood. Whether bladder tumor cells are destroyed by nonspecific mechanisms or targeted by specifically activated lymphocytes recognizing cognate antigens is unclear. To investigate a possible cross-reactivity between BCG and bladder cell tumors, we tested before BCG treatment the lymphoproliferation of peripheral blood lymphocytes against several mycobacterial antigens, including the secreted fibronectin binding antigen 85 complex from BCG (AG 85) in patients with superficial bladder tumors compared to control matched patients. MATERIALS AND METHODS: Using a whole blood assay, T cell response against purified protein derivative, BCG extract, whole BCG, purified AG 85, and the nonspecific mitogens pokeweed and phytohemagglutinin was investigated in 79 patients with superficial bladder tumors before BCG and in 39 control subjects without malignancy matched for age and sex. Neither group had a history of tuberculosis. Lymphoproliferation was measured with a tritiated thymidine uptake assay on day 7 of culture. RESULTS: Of the 79 patients with superficial transitional cell carcinoma, a significant lymphoproliferative response before BCG against PPD, BCG extract, whole BCG and AG 85 was observed in 65 (82.2%), 67 (84.81%), 30 (37.97%) and 49 (62.02%) patients, respectively. Of the 39 controls only 26 (64.1%), 23 (58.9%), 3 (7.7%) and 3 (7.7%) patients, respectively, had a significant lymphoproliferation against PPD, BCG extract, BCG and AG 85 (p >0.05, p = 0.004, p = 0.00001 and p = 0.00001, respectively). In terms of lymphoproliferative levels, patients with superficial transitional cell carcinoma also showed a significantly higher response against PPD (p = 0.000012), BCG extract (p = 0.000001), AG 85 (p = 0.000001), whole BCG (p = 0.00001) and pokeweed (p = 0.01) than controls but not against phytohemagglutinin. CONCLUSIONS: Patients with superficial transitional cell carcinoma demonstrate an increased lymphoproliferation against mycobacterial antigens before BCG compared to control subjects. Although a nonspecific activation of the immune system cannot be excluded at this stage, our data may suggest the possible existence of bladder cancer antigens cross-reactive with mycobacterial antigens responsible for boosting precursor cells witnessing previous contacts with mycobacteria. The implication of these findings in the antitumoral mechanism of action of BCG are under investigation.

Cross-reactive immune responses against Mycobacterium bovis BCG in mice infected with non-tuberculous mycobacteria belonging to the MAIS-Group.

Two bacillus Calmette-Guérin (BCG)-susceptible mouse strains, BALB/c and C57BL/6, were infected intravenously with Mycobacterium intracellulare, M. avium or M. scrofulaceum and monitored during 3 months for mycobacterial replication and antibody and Th1-type cytokine production in response to cytoplasmic and secreted antigens from M. bovis BCG. Whereas initial colony-forming unit (CFU) counts of M. intracellulare and M. avium were higher in lungs than in spleen, the opposite was observed for M. scrofulaceum. Mycobacterium intracellulare was the most virulent species and its replication could not be controlled in either mouse strain. It also induced the strongest antibody response. Mycobacterium avium was eliminated in both mouse strains and M. scrofulaceum finally was eliminated in C57BL/6 but multiplied in spleen from BALB/c mice. Significant sustained interleukin-2 and interferon-gamma production towards BCG antigens was only found in M. scrofulaceum infection. As in BCG-vaccination, M. scrofulaceum-infected C57BL/6 mice demonstrated a higher response towards whole BCG culture filtrate, BCG extract and purified antigen 85 complex (Ag85) from BCG than did BALB/c mice. The data suggest that the presence of M. scrofulaceum in the environment may possibly interfere in genetically predisposed subjects with BCG vaccine and its protective efficacy against M. tuberculosis.

Humoral response against heat shock proteins and other mycobacterial antigens after intravesical treatment with bacille Calmette-Guérin (BCG) in patients with superficial bladder cancer.

Few studies have analysed the antibody response during intravesical BCG immunotherapy for superficial bladder cancer. We have examined the evolution in serum antibody response against several heat shock proteins (hsp), including the recombinant mycobacterial hsp65 and the native protein P64 from BCG, GroEL from Escherichia coli (hsp60 family), recombinant mycobacterial hsp70 and the E. coli DnaK (hsp70 family), against purified protein derivative of tuberculin (PPD) and the AG85 complex of Mycobacterium bovis BCG, as well as against tetanus toxoid in 42 patients with a superficial bladder tumour, 28 treated with six intravesical BCG instillations and 14 patients used as controls. We also analysed the lymphoproliferative response of peripheral blood mononuclear cells against PPD in this population. Data of antibody responses at 6 weeks post BCG were available in all 28 patients, and at 4 month follow up in 17 patients. All patients who demonstrated a significant increase in IgG antibodies against PPD at 4 months follow up had a significant increase already at 6 weeks of follow up. In contrast, IgG antibodies against hsp increased significantly from 6 weeks to 4 months post-treatment. A significant increase in IgG antibodies against PPD, hsp65, P64, GroEL, and hsp70 at 4 months follow up was observed in 10/17, 8/17, 10/17, 4/17 and 8/17 patients. Native P64 protein elicited a higher antibody response than recombinant mycobacterial hsp65. No increase in antibody response was observed against Dnak from E. coli, against AG85 or tetanus toxoid after BCG therapy. An increase in IgG antibodies against P64 at 4 months follow up compared with pretreatment values was found to be a significant predictor of tumour recurrence (P<0.01). Further studies with a larger number of patients are needed to confirm the value of the antibody response against P64 as a clinical independent prognostic factor.

Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex.

BALB/c and C57BL/6 mice were injected intramuscularly with plasmid DNA encoding the three components of the immunodominant 30-32 kDa antigen 85 complex (Ag85A, Ag85B, and Ag85C) from Mycobacterium tuberculosis culture filtrate, in order to investigate the utility of nucleic acid vaccination for induction of immune responses against mycobacterial antigens. Ag85A and Ag85B encoding plasmids induced a robust Th1-like response towards native Ag85, characterized by elevated levels of interleukin (IL)-2, interferon-gamma, and TNF-alpha. Levels of IL-4, IL-6, and IL-10 were low or undetectable. Plasmid encoding Ag85C was not effective. Cytotoxic T cell activity was also generated in in vitro restimulated splenocyte cultures from Ag85A and Ag85B DNA vaccinated mice. Finally, Ag85A and Ag85B DNA vaccination conferred significant protection against mycobacterial replication in lungs from B6 mice, subsequently challenged. Therefore, this technique may be useful for the definition of protective antigens of M. tuberculosis and the development of a more effective tuberculosis vaccine.

Detection of rifampicin and isoniazid resistances of Mycobacterium tuberculosis strains by particle counting immunoassay (PACIA).

The particle agglutinated counting immunoassay (PACIA) was used to determine the susceptibility of Mycobacterium tuberculosis strains to the two major antimycobacterial drugs, isoniazid and rifampicin. On evaluating 12 M. tuberculosis strains with different sensitivities, our results were in complete accordance with those obtained using the well-known BACTEC system. The PACIA technique is automated and quite inexpensive. Interpretation of the test may be achieved in as little as five days.

[The need of prolonged BCG treatment in superficial bladder cancer is suggested by the development of a peripheral immune response induced by BCG]. / La nécessité d'un traitement prolongé par BCG dans les cancers superficiels de la vessie est suggérée par l'évolution de la réponse immunitaire périphérique induite par le BCG.

Optimal duration of immunotherapy treatment by BCG for the prevention of recurrences of superficial bladder cancer is still unknown. We have studied the evolution and duration of the cellular immunity response at the peripheral level after BCG intravesical instillations. Our results show that immunity activation after BCG is of short duration and don't take more than 6 months. Our results support, strengthen and partially allow to explain the utility of maintenance treatment by BCG following 6-weekly instillations.

Evolution and clinical significance of the T cell proliferative and cytokine response directed against the fibronectin binding antigen 85 complex of bacillus Calmette-Guerin during intravesical treatment of superficial bladder cancer.

PURPOSE: The antitumorigenic effect of intravesical bacillus Calmette-Guerin (BCG) in superficial bladder cancer was reported to be initiated by the attachment of BCG to the bladder wall via fibronectin. The antigen 85 complex secreted in BCG culture filtrate binds specifically to fibronectin and is a powerful T cell stimulus. Therefore, we investigated the evolution and clinical significance of the cellular proliferative response and cytokine production during intravesical BCG therapy against this purified antigen 85 complex. MATERIALS AND METHODS: Evolution of the lymphoproliferation, interleukin-2 and interferon-gamma production of peripheral blood lymphocytes against tuberculin (purified protein derivative), purified antigen 85, BCG culture filtrate, whole BCG bacilli and pokeweed mitogen was tested before and after 6 weekly intravesical BCG instillations in 29 patients with superficial bladder cancer at intermediate or high risk for recurrence. RESULTS: A major increase in the lymphoproliferative response against purified protein derivative, antigen 85, BCG culture filtrate, whole BCG and pokeweed mitogen was observed in 69.0, 65.5, 79.3, 48.3 and 65.3% of the patients, respectively, analyzed after BCG therapy. Reactivity returned to baseline values at 6 months of followup. Of the patients who received a second BCG course because of tumor recurrence 66% had a novel increase in lymphoproliferation against antigen 85. An increase in the production of interleukin-2 and interferon-gamma by peripheral lymphocytes against antigen 85 was noted in 42.1 and 50% of the treated patients, respectively, after a single BCG course. During a mean followup of 23.11 months 48.5% of the patients remained tumor-free. No correlation could be found between the immunological response against any of the BCG antigens and the clinical evolution of the response. CONCLUSIONS: Intravesical BCG instillations induce a transient (less than 6 months) peripheral immune activation against several purified BCG antigens and among them the fibronectin binding antigen 85 complex. Reactivation is observed in most cases after additional BCG courses. The absence of long lasting immune activation after a single 6-week course of BCG could be related to the increased clinical efficacy observed with BCG maintenance instillations.

Immunogenicity and protective efficacy of a tuberculosis DNA vaccine.

Tuberculosis is the most widespread and lethal infectious disease affecting humans. Immunization of mice with plasmid DNA constructs encoding one of the secreted components of Mycobacterium tuberculosis, antigen 85 (Ag85), induced substantial humoral and cell-mediated immune responses and conferred significant protection against challenge with live M. tuberculosis and M. bovis bacille Calmette-Guérin (BCG). These results indicate that immunization with DNA encoding a mycobacterial antigen provides an efficient and simple method for generating protective immunity and that this technique may be useful for defining the protective antigens of M. tuberculosis, leading to the development of a more effective vaccine.

Fibronectin-binding antigen 85 and the 10-kilodalton GroES-related heat shock protein are the predominant TH-1 response inducers in leprosy contacts.

Peripheral blood mononuclear cells from 27 healthy leprosy contacts were analyzed for lymphoproliferation and TH-1 cytokine secretion (interleukin-2 and gamma interferon) in response to heat shock proteins with molecular masses of 65, 18, and 10 kDa from Mycobacterium leprae and the 30-32-kDa antigen 85 (Ag 85) from Mycobacterium bovis BCG. Cells from 18 and 19 of 19 lepromin-positive contacts proliferated or produced TH-1 cytokines in response to the M. leprae 10-kDa protein and to Ag 85, respectively. Limiting-dilution analysis for two lepromin-positive contacts indicated that about one-third of M. leprae-reactive T cells displayed specificity to the M. leprae 10-kDa protein and Ag 85. The M. leprae 65- and 18-kDa proteins were less potent TH-1 response inducers: gamma interferon and interleukin-2 could be measured in 14 and 19 lepromin-positive contacts, respectively. In contrast, very low or undetectable proliferative and cytokine responses were found for 8 lepromin-negative contacts. Our data demonstrate that the fibronectin-binding Ag 85 and the 10-kDa GroES homolog are powerful mycobacterial TH-1 response inducers in the vast majority of lepromin-positive contacts and suggest that they might be valuable candidates for a future subunit vaccine.

IgG humoral response against the antigen 85 complex homologues in leprosy.

Antigen 85 complex is the major protein component present in M. bovis BCG culture filtrate (CF). It consists of a family of three proteins: 85A, 85B and 85C. Combining isoelectric focusing and Western blot analysis, we have previously identified different antigenically related proteins present in the CF of other mycobacteria (M. tuberculosis, M. kansasii, M. avium, M. gordonae, M. fortuitum and M. phlei) using monoclonal antibodies (MoAbs) directed against the antigen 85 complex of M. bovis BCG. Humoral immune response directed against these cross-reactive homologues was analysed in sera from 20 patients with multibacillary leprosy (BL/LL), from 20 patients with paucibacillary leprosy (BT/TT) and from 15 healthy leprosy contacts. All the antigen 85 homologues identified in the seven CFs by MoAbs were also recognized by IgG present in sera from multibacillary leprosy patients, but not or very faintly in sera from paucibacillary leprosy patients or from healthy subjects. These results suggest that some of the M. leprae epitopes inducing a significant humoral response in multibacillary leprosy are common to the various 85 antigenically related proteins present in all mycobacterial species.

Evolution of cellular and humoral response against Tuberculin and antigen 85 complex during intravesical treatment with BCG of superficial bladder cancer.

Prophylactic treatment with Bacillus Calmette-Guerin (BCG) is an established and effective therapy of bladder cancer. The antitumor effect of BCG seems to be largely related to cellular immunological mechanisms, although its precise mode of action is unknown. Antitumor response of BCG seems to be initiated by the attachment of BCG to bladder wall via Fibronectin (FN). The cellular immune response against Tuberculin PPD and the major secreted BCG antigen (Fibronectin-binding AG 85 complex) has been tested in a control group of 20 untreated bladder tumor patients and before and after 6 weekly intravesical BCG instillations in a group of 20 superficial bladder tumor patients. A major increase in the lymphoproliferative response against PPD and AG 85 was observed in respectively 66% and 57% of the treated patients. In contrast, no detectable antibody response (IgA, IgM, IgG) was observed against AG 85 complex after BCG treatment. On the other hand, antibodies against Tuberculin increased in 13 of 20 patients. This study seems to demonstrate a specific cellular immune activation against AG 85 Fibronectin-binding complex during BCG treatment of superficial bladder tumors. Humoral response against the AG 85 is not activated after BCG treatment. Further studies are needed to elucidate the role of AG 85 in the cellular intravesical penetration of BCG. Presence or absence of cellular response against this antigen could be of clinical value.

T-cell-epitope mapping of the major secreted mycobacterial antigen Ag85A in tuberculosis and leprosy.

Lymphoproliferation and gamma interferon (IFN-gamma) secretion in response to 28 overlapping 20-mer synthetic peptides covering the complete sequence of the mature (295-amino-acid) 85A component of the major secreted, fibronectin-binding antigen 85 complex from Mycobacterium tuberculosis and Mycobacterium bovis BCG (MTAg85A) was examined by using peripheral blood mononuclear cell (PBMC) cultures from healthy tuberculin- and lepromin-positive volunteers and from patients with tuberculosis and leprosy. Peptide recognition was largely promiscuous, with a variety of human leukocyte antigen haplotypes reacting to the same peptides. PBMC from all tuberculin-positive subjects reacted to Ag85, and the majority proliferated in response to peptide 6 (amino acids 51 to 70), peptides 13, 14, and 15 (amino acids 121 to 160), or peptides 20 and 21 (amino acids 191 to 220). PBMC from tuberculosis patients demonstrated a variable reactivity to Ag85 and its peptides, and the strongest proliferation was observed against peptide 7 (amino acids 61 to 80). MTAg85A peptides were also recognized by PBMC from healthy lepromin-positive volunteers and paucibacillary leprosy patients (again in a promiscuous manner), but despite a 90% homology between the 85A proteins of M. leprae and M. tuberculosis, the peptides recognized were different. PBMC from lepromin-positive healthy contacts reacted against peptide 2 (amino acids 11 to 30), peptide 5 (amino acids 41 to 60), and peptides 25 and 26 (amino acids 241 to 270). PBMC from paucibacillary patients reacted preferentially against peptide 1 (amino acids 1 to 20) and peptide 5. Multibacillary patients were not reactive to Ag85 or the MT85A peptides. IFN-gamma production was generally detected simultaneously with positive lymphoproliferative responses, although peptide 1 mostly stimulated proliferation and peptides 27 and 28 mostly elicited an IFN-gamma response. In conclusion, regions 41 to 80 and 241 to 295 demonstrated powerful and promiscuous T-cell-stimulatory properties, resulting in proliferative responses and IFN-gamma secretion, respectively, in the majority of reactive subjects tested in this study. These results could be of value in the development of a subunit vaccine for tuberculosis and leprosy.

T cell reactivity against antigen 85 but not against the 18- and 65-kD heat shock proteins in the early stages of acquired immunity against Mycobacterium leprae.

T cell proliferation and interferon-gamma (IFN-gamma) production of peripheral blood mononuclear cells (PBMC) from 20 household contacts were tested against the 18- and 65-kD heat shock proteins from Mycobacterium leprae (ML18 and ML65 respectively) and antigen 85 from Myco. bovis bacille Calmette-Guérin (BCG) (Ag 85) during a 12-months follow-up study. Among the eight contacts that became positive, eight showed positive reactivity against Ag 85, 5/8 against ML65 and 4/8 against ML18 at the end of the study. Of the 16 contacts who were lepromin-positive either at first or second testing, all responded to Ag 85, 11 to ML 65, but only eight reacted to ML18 antigen. Contacts who were lepromin-positive at first testing developed responses to ML18 only at second testing. In contrast, among the four contacts that remained lepromin-negative during the follow up, three proliferated to Ag 85 either at first or second testing, but only one produced IFN-gamma against Ag 85 at the end of the study. These results demonstrated that T cell reactivity and particularly IFN-gamma secretion against Ag 85, but not against ML18 and ML65, might be a predominant mechanism in the early stages of acquired protective immunity against Myco. leprae.

Structure of the Mycobacterium tuberculosis antigen 88, a protein related to the Escherichia coli PstA periplasmic phosphate permease subunit.

We report the cloning and sequencing of the gene coding for antigen 88 from Mycobacterium tuberculosis by using monoclonal antibodies to screen an expression library in lambda gt11. The gene encodes a 403-amino-acid-residue protein with a calculated molecular mass of 43,790 Da which contains seven putative transmembrane alpha-helical domains and presents a significant homology to the PstA protein of Escherichia coli. In its N-terminal region, it contains a 61-amino-acid region highly homologous to the fifth transmembrane helix of E. coli PstC. PstA and PstC are the two hydrophobic subunits of an E. coli periplasmic phosphate permease. Since the phosphate-binding subunit of this putative permease in M. tuberculosis has previously been characterized, i.e., the 38-kDa mycobacterial protein (also called protein antigen b, Ag 5, and Ag 78) homologous to PstS of E. coli, it seems likely that functional permeases analogous to the periplasmic permeases of gram-negative bacteria also exist in mycobacteria.