Alfred J. Crowle

Chlorpromazine: A Drug Potentially Useful for Treating Mycobacterial Infections

Chlorpromazine (CPZ) is one of several phenothiazines known to have antimicrobial properties. It can inhibit mycobacteria, and was reported in the early literature to improve tuberculosis clinically. CPZ was tested here for its ability to inhibit the replication of Mycobacterium tuberculosis and Mycobacterium avium in cultured normal human macrophages, as determined by counts of viable bacteria at 0, 4, and 7 days after bacterial infection of the macrophages. CPZ inhibited the intracellular bacteria at a concentration range of 0.23-3.6 micrograms/ml, and was more effective intracellularly than extracellularly. It was further tested for its ability to cooperate with isoniazid, streptomycin, pyrazinamide, rifampin, rifabutin, penicillin and ethambutol (EMB) against intramacrophage M. tuberculosis and M. avium. CPZ enhanced the effectiveness of most of the drugs tested against intracellular mycobacteria. However, the combination of CPZ and EMB did not result in augmented antimycobacterial activity.

An improved stain for immunodiffusion tests

We describe an improved immunodiffusion stain using trichloroacetic acid to mordant dying by Crocein scarlet. Background is readily cleared without over-destaining using 0.3% acetic acid.

Combined effect of pyrazinamide and ofloxacin within the human macrophage.

SETTING Recent reports of outbreaks of multidrug resistant tuberculosis have raised questions as to the most appropriate therapeutic response for those exposed to such organisms. A recent Centers for Disease Control National Action Plan suggests the combination of pyrazinamide (PZA) and a quinolone as a potential preventive therapy regimen. OBJECTIVE Prior studies in the ex-vivo human macrophage model have shown PZA to have only a bacteriostatic effect and, in addition, to diminish the bactericidal effect of rifampin. This study was designed to quantify the intramacrophage antimycobacterial effect of PZA when combined with a quinolone (ofloxacin). DESIGN Forty micrograms/ml of PZA was combined with varying concentrations of ofloxacin and administered to human macrophages infected with virulent tubercle bacilli; drug sequencing was also studied. RESULTS A clinically achievable level of PZA enhances the antimycobacterial effect of low, non-bactericidal levels of ofloxacin and does not impede the bactericidal effect of a higher clinically effective level of ofloxacin. Unlike the combination of PZA and rifampin, these interactive effects are not affected by the sequence of drug administration. CONCLUSIONS Findings support the use of these agents as a potentially effective preventive therapy combination for individuals exposed to multidrug resistant tuberculous organisms.

The Tubercle Bacillus­ Human Macrophage Relationship Studied In Vitro

Tuberculosis is an infection of the monocytic phagocyte (MP) system.1–8 The disease-causing bacilli multiply in cells of this system as determined by bodily environment. For example, they reproduce freely in alveolar macrophages (MP) at the beginning of human infection because these cells lack resistance, are located in oxygen-rich areas that support the oxygen needs of these aerobic bacteria, and are not yet receiving signals for immune activation from the body’s T-cell system.4,9–11 But later, the bacteria are inhibited by MP, for instance, in lymph nodes, because these cells are in oxygen-poor areas and are being defensively activated by immune lymphokines that T cells responding to bacillary antigens have begun to produce.4,7–9,12 The MP may also respond detrimentally to infection-exacerbating molecular signals, like γ-globulin produced by tuberculoprotein-hypersensitive lymphocytes.5,7–9,13 Then, they actively promote bacillary growth.

Effects of isoniazid and of ceforanide against virulent tubercle bacilli in cultured human macrophages.

Isoniazid (INH) is said to inhibit tubercle bacilli equally well in vivo and in vitro, and to be mycobactericidal. Ceforanide (CEF) can inhibit tubercle bacilli in vitro but has been found ineffective clinically. These two drugs were tested against virulent tubercle bacilli in cultured human macrophages (MP), partly to compare the results with clinical experience, and partly for a better understanding of antituberculosis activities of these drugs in human beings. INH had the same minimal inhibitory concentration (MIC) against tubercle bacilli in MP as in 7H9 broth cultures. It killed multiplying bacilli in MP but not nonmultiplying bacilli, even at 100 times MIC. It killed both multiplying and nonmultiplying bacilli in broth cultures. It interfered with its own effectiveness against intra-MP bacilli by preventing nonmultiplying bacilli from beginning to multiply and thus become susceptible to killing. These findings help explain why this demonstrably mycobactericidal drug produces relapses of tuberculosis when used alone. It was confirmed that CEF is able to inhibit growth in broth cultures (MIC = 10 micrograms/ml). However, it was not effective against either multiplying or nonmultiplying bacilli in MP at concentrations up to 50 micrograms/ml. These results with the drugs INH and CEF support the good record of correlation between the human MP model of tuberculosis and clinical experience in antituberculosis chemotherapy.

Crossed immunoelectrophoresis of rat serum

A crossed immunoelectrophoresis (X-IEP) reference profile of normal (Sprague-Dawley) rat serum antigens was developed. Twenty-three antigens were detected; 13 were identified. These included inter-alpha-trypsin inhibitor, alpha 1-antichymotrypsin, and fibronectin, which had not previously been identified in rat serum. Many of the antigens were similar electrophoretically to their analogs in human and mouse serum, but some were substantially different. Inter-alpha-trypsin inhibitor, fibronectin, and C3 globulin were more anodic, while transferrin was more cathodic. Rat haptoglobins mobility was between those of human and mouse haptoglobins. As in the mouse, but more so, alpha-macroglobulin in the rat had alpha 1 mobility rather than alpha 2 seen for this protein in human serum. Only rat alpha 1-antichymotrypsin exhibited a double peak. There were variations in haptoglobin and alpha 1-lipoprotein mobilities among different strains of rat. This establishment of a standard X-IEP map of rat serum will simultaneously permit multiple quantitative and qualitative analyses of rat serum antigens for various experimental and clinical purposes.

Mechanisms involved in the antibody-mediated suppression of tuberculin-type delayed hypersensitivity: II. The sensitivity of tolerance induction to cyclophosphamide pretreatment and splenectomy, and the demonstration of active suppression

Abstract The induction of tuberculin-type delayed hypersensitivity, as measured by skin test, can be specifically inhibited by administration of antibody during sensitization. The cellular mechanisms involved in this tolerance were investigated in CAP 1 mice, using chicken conalbumin as antigen. Tolerance was prevented when mice were treated with Cyclophosphamide 2 days before sensitization and suppression. However, it was not affected by splenectomy 7 or 21 days before sensitization. This tolerance could be transferred to normal CAF 1 mice with spleen cells, but not with thymocytes, when taken from donor mice 21 to 28 days after sensitization and tolerance induction. Production of these cells in the donors required both antibody and antigen. The cells responsible for the transfer were B cells, as shown by their sensitivity to rabbit anti-mouse-immunoglobulin serum and complement. In addition to B cells, serum from tolerant mice also could transfer suppression at 21 to 28 days. We conclude that sensitizing mice, and treating them with specific immunosuppressive antiserum, induces the recipients to make suppressor B cells and suppressive humoral factors, which are involved in arresting the induction of tuberculin-type delayed hypersensitivity.

Differential serum protein binding of benzidine- and benzidine-congener based dyes and their derivatives

Environmental dyes and their derivatives, some of which are genotoxic, must be transported within the body to the tissues which they affect. One mechanism for this can be observed directly by crossed immunoelectrophoresis (X-IEP). Binding of these chemicals to certain serum proteins changes electrophoretic and immunoprecipitation morphology in X-IEP patterns. This is demonstrated here for four azo dyes derived from benzidine, 3,3′-dimethylbenzidine, and 3,3′-dimethoxybenzidine, and their parent aromatic amines. Direct Red 2 (a 3,3′-dimethylbenzidine-based dye), Direct Blue 15 (a 3,3′-dimethoxybenzidine-based dye), Direct Black 38 (a benzidinebased dye), and Evans Blue (a 3,3′-dimethylbenzidine-based dye) all bound to albumin, α1-lipoprotein, β-lipoprotein, and hemopexin. Direct Red 2 only slightly affected the mobilities of these proteins. Direct Blue 15 bound also to prealbumin and α1-antichymotrypsin, and degraded C3 globulin. Direct Black 38 and Evans Blue bound to numerous additional proteins. Evans Blue bound variably to proteins of sera from different individuals, suggesting that there are individual differences in serum protein binding capabilities for these chemicals. Of the three derivatives of the benzidine dyes, only 3,3′-dimethylbenzidine caused changes in X-IEP patterns, indicating its binding to the serum proteins. This chemical differentially affected sub-populations of α1-lipoprotein, either by altering its electrophoretic mobility or inhibiting its recognition by antibodies. Autoradiographic analyses demonstrated the binding of benzidine and 3,3′-dimethylbenzidine to both α1- and β-lipoproteins.

Effects of aging in A/J mice on delayed and arthus hypersensitivities, anamnesis and a low-dose tolerance.

This paper describes a 2-year experiment on the flux with aging in A/Jax female mice of Arthus and tuberculin-type delayed hypersensitivities to chicken conalbumin antigen, of delayed hypersensitivity

Anaphylactic and precipitating antibody responses of aging A/J female mice.

Aging A/Jax female mice were compared with young controls for abilities throughout life to make IgG1 and IgE anaphylactic antibodies and precipitins to chicken conalbumin (CA) and methylated human serum albumin (MeHSA) injected in five different regimens of immunization. These regimens consisted of various combinations of injections of antigen in water-in-oil emulsion or in saline. CA induce those antibodies well, MeHSA poorly. The mice were able to respond nearly equally at all ages, a finding differing from earlier ones for at least two reasons: (1) neither antigen used is related to any in mouse food, and (2) the regimens of immunization caused sustained antigenic stimulation together with induction of T cell responses, which could have compensated for otherwise expected age-weakening of necessary TH cell functionings. We conclude that A/J female mice can make good anaphylactic and precipitating antibody responses to new antigens throughout life.