J.P. Van Vooren

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.

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.

Constitutive release of metalloproteinase-9 (92-kd type IV collagenase) by Kaposi's sarcoma cells

Kaposis sarcoma (KS) is an angioproliferative disease characterized by proliferating spindle-shaped cells, angiogenesis, and inflammatory cell infiltration. Several lines of evidence suggest that KS is a multifocal cytokine-mediated disease of vascular origin. Because metalloproteinases (MMPs) are important enzymes involved in angiogenesis, we studied their activity in five different KS-derived cell lines and compared these data with those obtained with human umbilical vein endothelial cells (HUVEC). We focused on the activity of the 72- and 92-kd type IV collagenases because these enzymes are thought to play an important role in the process of tumoral invasion. Nonstimulated HUVEC released a weak 72-kd collagenase activity and no 92-kd collagenase activity, as determined by zymographic analysis. Stimulation of HUVEC with phorbol myristate acetate (PMA) or TNF-alpha increased the 72-kd collagenase activity and also induced a 92-kd collagenase activity. By contrast, KS-derived cells constitutively released significant 72- and 92-kd collagenase activities. The basal release of these enzymes by KS cells was further enhanced by TNF-alpha or PMA. Conversely after in vivo exposure to chemotherapy, KS-derived cells showed a downregulation of the production of MMPS that could be reversed by the addition of TNF or PMA. These results suggest that KS cells have constitutive features of activated cells that have an invasive and metastasizing potential.

Rapid detection of tuberculous and non-tuberculous mycobacteria by polymerase chain reaction amplification of a 162 bp DNA fragment from antigen 85.

A polymerase chain reaction (PCR) assay was developed for detection of mycobacteria using amplification of a 162 bp region of the genes coding for the mycobacterial antigen 85 complex. Strains belonging to theMycobacterium tuberculosis complex were further differentiated from non-tuberculous mycobacteria by hybridization of the PCR derived Southern blot with an internal oligonucleotide probe and washing under stringent conditions. The method allowed rapid and sensitive detection of mycobacterial DNA in uncultured clinical samples. PCR results obtained forMycobacterium tuberculosis in 206 specimens from 180 untreated patients gave a sensitivity of 93.9% and a specificity of 94.3% compared with the culture. PCR detected DNA fromMycobacterium tuberculosis in seven samples from patients with clinically evident tuberculosis in whom culture was negative. The results suggest that this PCR assay could be used for early and specific diagnosis of tuberculosis.

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‐γ 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.

Effect of zinc deficiency on the appearance of two immunodominant protein antigens (32 kDa and 65 kDa) in culture filtrates of mycobacteria

After growth of six strains of mycobacteria on Sauton medium in the absence of added Zn2+, cell yields were lowered, to between 22% and 67% of the yields obtained when Zn2+ (5 microM) was added. Two immunodominant proteins, named P64 and P32 (antigens of 62-65 kDa and 29-33 kDa, respectively) were abundant in culture filtrates after growth of mycobacteria. P64 was present at elevated concentrations (showing a 9- to 16-fold increase as a percentage of the total protein released) after Zn2+-deficient growth of five of the six strains studied; in Mycobacterium tuberculosis it represented 25% of all released proteins. However, little P64 was detected in culture filtrates of M. fortuitum and of M. phlei grown under Zn2+ deficiency, and in the latter there was no increase of P64 during Zn2+ deficiency.

A multidot immunobinding assay for the serodiagnosis of tuberculosis: Comparison with an enzyme-linked immunosorbent assay

A simple dot immunobinding (dot blot) assay procedure has been developed for the detection of antibodies directed against a soluble mycobacterial antigen preparation. This technique was compared with the widely used ELISA, in a study of samples from tuberculous patients. Dot blots were read on a densitometer. The correlation between both assays was excellent (r = 0.91; P less than 0.001); 90% of sera from tuberculous patients were detected using both techniques and a serial two-fold dilution method. Assessments of the end-points of titration curves by reflectometry and simple visual interpretation gave similar results. The dot blot assay is easier to perform and appears to be a practical alternative to ELISA for the detection of anti-mycobacterial antibodies in tuberculous patients.

Detection of fastidious mycobacteria in human intestines by the polymerase chain reaction.

The aim of this study was to determine whether difficult-to-grow mycobacteria are present in human intestines. Intestinal tissue samples were subjected to both mycobacterial culture and a polymerase chain reaction (PCR) assay. After detection by PCR, species identity was determined by hybridizing the amplified 16S rRNA gene fragments with species-specific oligonucleotides. Intestinal biopsies from 63 patients with noninflammatory bowel diseases (n=22), Crohns disease (n=31), or ulcerative colitis (n=10) were analyzed. Culture and PCR revealed mycobacteria in four (6%) and 25 (40%) samples, respectively. Samples positive by PCR were negative with all probes specific to nine common cultivable species but were positive with theMycobacterium genavense-specific probe in 68% of cases. Mycobacterial isolates were identified asMycobacterium gordonae andMycobacterium chelonae. Findings were similar in Crohns disease samples compared to non-Crohns disease samples. This study shows that difficult-to-grow mycobacteria can be detected by PCR in large and similar proportions of inflamed intestinal tissue from patients with inflammatory bowel disease and intestinal tissue that appears normal from patients with noninflammatory bowel disease.

Treatment of Kaposi's sarcoma with human chorionic gonadotropin.

Clinical-grade preparations of human chorionic gonadotropin (hCG) have been shown to be toxic to Kaposi’s sarcoma (KS) cells. However, the mechanism of the anti-KS activity achieved with these preparations remains unclear. The results of clinical studies using commercial hCG preparations in human KS are also highly contradictory. The apparent controversy between different studies may be due to the fact that pro- and anti-KS components are present in varying proportions in different hCG preparations. As certain hCG preparations could not only lack the ability to control KS but also contain some contaminant KS growth factor(s), we suggest a cautious use of crude hCG for the treatment of KS.

Phenotypic characteristics of Kaposi's sarcoma tumour cells derived from patch-, plaque- and nodular-stage lesions: analysis of cell cultures isolated from AIDS and non-AIDS patients and review of the literature: KAPOSI's SARCOMA PHENOTYPE

Background  Kaposis sarcoma (KS) is commonly thought to be derived from endothelial cells because of the predominant expression of endothelial markers in KS lesions. However, the heterogeneity of the spindle‐cell compartment makes the precise lineage relationship of KS tumour cells unclear. Cultured KS‐derived spindle cells constitutively overexpress antiapoptotic proteins and exhibit invasive properties, which suggests that they may adequately represent the tumour cells of KS. Objectives We aimed to investigate the expression of a wide variety of immunohistochemical markers by spindle cells derived from patch‐, plaque‐ and nodular‐stage lesions from patients with iatrogenic, sporadic and acquired immune deficiency syndrome‐related KS, and to review the data reported by other laboratories. Methods Cells from six KS cell cultures derived from four subjects were examined by immunostaining. Results Comparison of these data indicates that KS‐derived spindle cells generally express myofibroblast antigens but lack endothelial and/or leucocyte markers. Conclusions As the myofibroblast phenotype is not the predominant feature of KS tissues, our findings further substantiate the view that the in vivo dominant endothelial population represents a reactive hyperplasia rather than the true KS tumour process.