Effect of Corynebacterium parvum on the response to irradiation of a C3H fibrosarcoma.

A methylcholanthrene-induced fibrosarcoma of C3H mice has been used as 5th- to 6th-generation syngeneic transplant in an investigation of the role of Corynebacterium parvum as an adjuvant to the therapeutic effect of local irradiation of established tumors. The most effective route for administration of C. parvum in this tumor system was i.v., and the greatest effect of the C. parvum-mediated tumor graft rejection was observed for tumor growing intracutaneously or s.c. An intermediate level of effectiveness was obtained for tumor growing i.m. The combined C. parvum and local irradiation studies were performed using tumors growing in the leg muscle and measuring 8 mm in diameter at the time of local irradiation. Several routes of administration of C. parvum, dose levels of C. parvum, and time relationships between administration of C. parvum and irradiation were investigated. The outstanding finding was that very low doses of radiation were quite effective when administered to tumors growing in animals pretreated with C. parvum. This was true for radiation administered as a single or fractionated dose (10 equal doses spread over 18 days). For single-dose irradiation the effect was relatively dose independent over the range of 200 to 3000 rads. Some indication was obtained that local irradiation may impair the tumor graft rejection reaction. No evidence was obtained of an enhanced growth of tumor. Regression of tumor following irradiation was not modified by pretreatment with C. parvum. The mice that were unsuccessfully treated by radiation and C. parvum had a lower incidence of metastatic disease in the lung than did the mice treated unsuccessfully with radiation alone.

Quantitative studies on the transplantability of murine and human tumors into the brain and subcutaneous tissues of NCr/Sed nude mice.

The transplantability of experimental tumors into the brain (i.c.) and s.c. tissues of C3Hf/Sed and athymic NCr/Sed nude mice was examined using quantitative cell transplantation assays. Studies using the immune-competent C3H animals showed that brain is a more favorable site for the transplantation of syngeneic tumor than s.c. tissue and that this is true for nonimmunogenic as well as immunogenic tumors. The capacity of the brain to act as an immunological sanctuary can be overwhelmed by a strong, systemic, secondary immune response such as that evoked by the methylcholanthrene-induced sarcoma FSal. In studies performed using NCr/Sed nude mice, the allogeneic tumor MCaIV was found not to be demonstrably immunogenic. The cell dose required to transplant the tumor into 50% of recipients (TD50) could neither be increased by immunization procedures nor decreased by six Gy whole-body irradiation (WBI) prior to transplantation. Delayed-type hypersensitivity to this tumor was not expressed by nude mice after rechallenge with tumor antigen. The TD50 was again lower for i.c. than s.c. transplantation and the ratio s.c./i.c. was comparable to that found in syngeneic C3Hf/Sed hosts. Three human tumors have been similarly tested. They were: FaDu, a pharyngeal squamous carcinoma; HFSal, a fibrosarcoma; and U87, a malignant glioma. s.c. TD50 values were in all cases significantly higher than those obtained i.c. The ratios TD50 s.c./i.c. ranged from 6.4 to greater than 50 in five studies, substantially higher than those found for transplantation of murine tumors into either the syngeneic or the allogeneic recipients. Six Gy WBI reduced the s.c. TD50 for these tumors, but in each case the value remained significantly higher than that obtained i.c. 19.4 Gy WBI given in 10 equal fractions and followed by i.v. bone marrow rescue reduced further the s.c. TD50 for FaDu. NCr/Sed nude mice demonstrated cross-reacting delayed-type hypersensitivity against FaDu and HFSal. A small proportion of FaDu tumors (less than 2%) displayed a spontaneous halt in growth or even regression. When the host cell infiltrate of these tumors was analyzed, an increase was seen in the proportion of Thy 1.2 and asialo-GM1-positive cells as compared with progressively growing tumors. These data strongly suggest that a residual low level of immune reactivity exists in nude mice against xenotransplanted human tumors. This resistance to s.c. transplantation may be diminished by WBI and is less for intracerebral implantation.

Acute graft-versus-host reaction in mice. 3. Organ distribution of injected 51 chromium labeled lymphocytes.

The distribution of labeled lymph node cells, causing an acute GvH reaction in lethally irradiated allogeneic recipients, was studied. Lymph node cells of C57BL mice were labeled with 51Cr and injected into lethally irradiated: a) C57BL mice, b) CBA mice, c) CBA mice sensitized to C57BL antigens prior to irradiation, d) CBA mice splenectomized before irradiation. Two more experimental situations were studied in which C57BL donors of lymph node cells were: e) presensitized to CBA antigens, or f) deprived of T-lymphocytes. The amount of radioactivity was determined in the whole body, blood, liver, spleen, subcutaneous lymph nodes, lungs, femora and kidneys of the irradiated recipient at regular intervals from the time of injection to the 120th hour after it. We found that living cells lodged predominantly in the spleen and the lymph nodes, while dead and dying cells accumulated in the liver. Other organs contained very small amounts of radioactivity. All the results point to the primary role of the spleen in the acute graft-versus-host reaction.

Chimaerism in lymph nodes of F1 into irradiated parental recipient chimaeras rejecting skin allografts from the other parent.

Mice of the C57BL strain were irradiated with 800 R over the whole body. The next day they received i.v. a mixture of 50 x 10(6) spleen and bone marrow cells from (C57BL x CBA-T6T6)F1 hybrid mice, and were challenged with CBA-T6T6 skin grafts later on. About 20% of the recipients rejected the CBA-T6T6 skin, whereas the others were completely tolerant for more than 200 days. By using the cytotoxic test, we found that both tolerant and nontolerant recipients were complete chimaeras, i.e., had only (C57BL x CBA-T6T6)F1 cells in their lymph nodes. However, analysis of the same mice by the chromosome marker technique disclosed a proportion of host (C57BL) cells in lymph nodes of both tolerant and nontolerant chimaeras. The percentage of host metaphases in nontolerant chimeras was significantly higher than that in tolerant chimaeras (P less than 0.01). It is possible therefore that the CBA-T6T6 skin grafts were rejected by residual host (C57BL) cells rather than (C57BL x CBA-T6T6)F1 cells reacting against skin-specific transplantation antigen(s) of the parental graft.

Inhibition of human monocyte spreading. An in vitro test for immunosuppressive potency of antihum lymphocyte globulin.

Monocytes from human peripheral blood, when incubated in vitro, spread onto the surface of the glass. Horse antihuman lymphocyte globulin (ALG) added to the incubation chamber inhibits the spreading, while normal horse globulin (NHG) does not. The inhibition depends on the concentration of ALG admixed to the mononuclear blood cells. Eleven coded samples of antihuman ALG were assayed for the ability to inhibit monocyte spreading. This potential was then compared to the in vivo immunosuppressive effect of the same samples determined by the prolongation of skin allograft survival in subhuman primates. It was found that the in vitro inhibitory acttivity correlated rather well with the in vivo immunosuppression, Therefore, the inhibition of monocyte spreading is proposed as an additional test system for the in vitro evaluation of the immunosuppressive potential of antihuman ALG.

Acute graft-versus-host reaction in mice. I. Cellular events.

Cellular events in the spleen during the development of acute GvH reaction in lethally irradiated recipients of allogeneic lymphocytes may be divided into 6 interrelated processes: (1) entering and lodging in the spleen of about 17% of inoculated cells which forms the compartment of potentially reactive cells; (2) transformation (recruitment) of about 30% of cells lodged in the spleen into large pyroninophilic cells (LPC), a process lasting about 12-16 hr; (3) proliferation of recruited LPC by five successive divisions with a Tc of about 12 hr; (4) transformation of LPC into non-LPC; (5) proliferation of non-LPC by one division, with a Tc of about 8 hr; and (6) migration of mature immunologically active cells from the spleen. The last process correlates well with the concomitant appearance of lymphocytes in the peripheral blood (killer cells) and with the time of acute death among inoculated mice.

The effect of allogeneic presensitization on Hya-isograft survival.

The effect of allogeneic presensitization on Hya isograft survival was reinvestigated in C57BL, C3H and (C57BL x C3H)F1 females. Three weeks after transplantation of H-2 or non-H-2 differing female or male skin grafts, a syngeneic male skin graft (Hya isograft) was transplanted onto female recipients. C57BL and (C57BL x C3H)F1 females pretreated with a female skin graft of any origin rejected the C57BL male isograft as a first-set, while females pretreated with a male skin graft rejected the C57BL male isograft in a second-set manner. The same happened with the C3H male skin graft transplanted onto previously challenged (C57BL x C3H)F1 females. C3H females pretreated with a C3H or CBA female graft rejected the C3H male isograft as a first-set, whereas those pretreated with a C3H or CBA male graft rejected the male C3H isograft in a second-set manner. However, after pretreatment with a BALB/c male skin graft, C3H females were not sensitized to Hya. They rejected the C3H male isograft slightly faster than was expected for the first graft but this accelerated rejection was not specific, since it occurred as well in C3H females pretreated with grafts of BALB/c females. The best explanation for this unsuccessful immunization of C3H females is that an inferior, non-H-2-dependent Hya recognition system is involved in male isograft rejection.

Phagocytic function in patients with recurrent urinary tract infections.

Phagocytes play an essential role in the host's defence against uropathogenic bacteria which are mostly extracellular pathogens. The functional capacity of peripheral blood phagocytes (predominantly polymorphonuclears, PMN) was investigated in 47 patients (urethrocystitis, acute or chronic pyelonephritis) and in healthy persons. The random mobility of phagocytes was determined by measuring their spontaneous migration from a capillary tube. Using radioactively labelled sheep erythrocytes as targets and the phagocytes as effector cells, ingestion, digestion and extracellular cytotoxicity were determined. All the four phagocytic functions in patients were significantly lower than in healthy controls, especially in patients with chronic pyelonephritis. These results link reduced phagocytosis by blood phagocytes with recurrent urinary tract infections (UTI). Whether the defect is primary or secondary to infection (and only transient) should be the object of further studies.

Papers published by members of the Croatian Academy of Medical Sciences, registered in international bibliographic data bases in the period 1986-1990.

Scientific productivity of the CAMS members was analyzed according to the number of papers published in periodicals covered by international Medline and Science Citation Index data bases from 1986 to 1990. Results showed the mean scientific productivity per CAMS member, and the relationship between scientific productivity and age, field of medicine or respective biomedical discipline. The pattern of scientific productivity was also found to change, when analyzed according to Medline or SCI data bases in separate.