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.

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.

Defective phagocytosis in chronic trichophytosis.

Chronic trichophytosis as a primary clinical entity (primary chronic trichophytosis, PCT) is increasingly encountered in the literature. Its prevalent cause in Croatia is the anthropophilic form of Trichophyton interdigitale. Chronicity of the disease may result from immunological defects of the patients. Thus, in 62 patients with PCT: skin testing, enumeration of T- and B-lymphocytes in the peripheral blood, quantitation of immunoglobulin classes, and phagocytosis (random mobility, ingestion, digestion and extracellular killing) by peripheral blood leukocytes was tested. The findings were compared to those in healthy persons. Defective phagocytosis (random mobility, ingestion and digestion) was found in patients with PCT (P < 0.001). All the other results were within the control range. Therefore, PCT seems to be associated with defective phagocytosis of peripheral blood leukocytes in affected persons.

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.

A comparative study on the xenotransplantability of human solid tumors into mice with different genetic immune deficiencies.

These experiments set out to assess the role of NK and B cells in the resistance of nude mice to human tumor xenotransplantation. The transplantability of 9 fresh and 8 cultured human tumors was compared in 2 strains of mice with different genetic immune deficiencies: athymic NCr/Sed (nu/nu) nude mice, and nude-beige-xid (N:NIH-nu-bg-xid/Sed mice). Flow cytometric studies showed both strains to be deficient in Thy. 1.2 (T) cells and unresponsive to stimulation by Concanavalin A (Con A) or direct T-cell-receptor triggering with anti-CD3. The number of B cells was similar in the 2 strains, but the response to lipopolysaccharide (LPS) was markedly reduced in the nude-beige-xid animals. The number of asialoGM1-positive cells (predominantly NK) detected by flow cytometry was also reduced in the nude-beige-xid mice. The transplantability of the human tumors was found to be equivalent in the 2 strains. Quantitative cell-transplantation assays performed for 2 of the tumor cell lines did not reveal any subtle transplantation advantage for the more broadly immune-deficient animals. No evidence could, therefore, be found to suggest that NK or B cells were major determinants of human tumor xenotransplantability in these strains of mice.

Residual immunity of athymic NCr/Sed nude mice and the xenotransplantation of human tumors.

This study assessed the residual immunity possessed by NCr/Sed (nu/nu) athymic nude mice and examined strategies to reduce it in order to enhance the transplantability of human tumors for experimentation. Adult (8-week-old) female mice had fewer T cells (11%) and more B and NK (asialo-GM1-positive [ASGM1+]) cells in their spleen than euthymic (nu/+) controls. The number of phenotypically mature T cells increased with age, peaking at 16 weeks. ASGM1+ cells also increased in number over time, although the NK-activity decreased after 12 weeks. B cells remained relatively constant in number. Athymic NCr/Sed nude mice displayed reactivity against a human squamous carcinoma xenograph (FaDu), in a Winn's test and TD50 assay. Immunity against xenografts (TD50 assay) was significantly lower (by a factor of 2) in 4-week-old than in 12-week-old nude mice. Similarly, a significant 2-fold reduction in TD50 was obtained after a single intraperitoneal injection of cyclophosphamide into 8-week-old animals. Chronic (greater than 8 weeks) exposure of the nude mice to subcutaneously administered beta-estradiol markedly reduced the number of splenic NK cells and their cytolytic activity, but the TD50 reduction was not statistically significant (p = 0.1). Six Gray whole-body irradiations (WBI) had been shown to produce a highly significant, 3-fold reduction in the TD50 for FaDu. Flow cytometric analysis of splenic lymphoid cells from whole-body-irradiated recipients revealed: (a) marked initial depletion in the absolute numbers of lymphoid cells; (b) marked and long-lasting depletion of T cells, with slow and minimal recovery only evident between 6 and 12 weeks; (c) rapid, almost complete, depletion of B cells with prompt and partial recovery after 2 weeks; (d) depletion of NK cells and NK activity, with recovery by 10 weeks. No change in the number or phagocytic capacity of resident peritoneal macrophages was seen. These data give further support to a postulated role for residual T cells in the xenoreactivity of NCr/Sed nude mice.

Quantitative transplantation assays of the rat rhabdomyosarcoma BA1112 isografts into the WAG/Rij Y rat and xenotransplantation into the athymic NCr (nu/nu) nude mouse.

Quantitative tumor cell transplantation assays have been performed to compare the transplantability of rat rhabdomyosarcoma BA1112 into isologous WAG/Rij Y rats and athymic NCr(nu/nu) nude mice. The end-point was the TD50 or the number of viable tumor cells which would transplant the tumors into half of the recipients. At Yale, two sets of 2-fold dilutions were prepared, one was sent to the MGH by Air Express. That afternoon, concurrent assays were performed at Yale using the WAG/Rij Y rat and at MGH using the NCr(nu/nu) mouse. The TD50 values were the same for iso- and xenotransplantation. Furthermore, the TD50s in rats and mice were unaffected by standard immunization procedures prior to challenge of the TD50 assay. The BA1112 (10(7) trypan blue excluding cells) grew to 10-12 mm and then completely regressed if transplanted into NCr(nu/+) mice which had received 6 Gy whole body irradiation but did not grow in control NCr(nu/+) mice. The times for the BA1112 to grow to 10 mm were the same in normal or preimmunized WAG/Rij Y rats or NCr(nu/nu) mice and in 6 Gy WBI NCr(nu/nu) mice. All of the experimental data show that the xenogenic NCr(nu/nu) mice accept the BA1112 as readily as do the isologous WAG/Rij Y rats.

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.

A peptidoglycan monomer as an antitumor agent in mice: stimulation of phagocytosis by resident peritoneal macrophages with peptidoglycan monomer.

Peptidoglycan monomer (dissacharide-pentapeptide, PGM) from Brevibacterium divaricatum is an immunomodulator and an antitumor agent. As part of our investigation of the antitumor properties of PGM in mice, its potential to stimulate phagocytosis by resident peritoneal macrophages was assessed. Inbred CBA/H/Zg tau mice (kept under conventional conditions) were used, and a simple and brief method was developed to evaluate phagocytosis. It consisted of a short (10 min) coincubation of yeast cells (YCs) with peritoneal cells (PCs) and microscopic (phase-contrast) scoring of YC ingestion. Three samples of PGM were injected intravenously into mice (60 mg/kg), and PCs were collected from groups of recipients 8, 16, 24, 48, or 72 h later. All samples temporarily increased phagocytosis, but the extent and duration of the effect varied. Stimulation of phagocytosis also occurred after in vitro exposure (3 h) of PCs to PGM, or to the pentapeptide (PP) part of its molecule. We concluded that PGM could enhance phagocytosis by resident peritoneal macrophages in vivo and in vitro, the PP being the active component in vitro.

In vitro inhibition of macrophage spreading by antigens and lymphokines: correlation with the footpad test and kinetics of inhibition.

As inhibition of spreading of mouse peritoneal macrophages is the basis for an in vitro test of cellular immunity, this test was used to investigate the correlation between in vitro and in vivo results and the kinetics of inhibition of spreading. BALB/c and NIH strain mice were immunized with human or bovine serum albumin or bovine gamma globulin. They displayed a positive footpad test when challenged with the specific antigen (21 days later). Peritoneal cells (PC) of mice with a strong footpad reaction were used for the spreading inhibition test. Intensity of spreading inhibition correlated well (r = -0.93) with that of the footpad reaction. Spreading inhibition had a cyclic pattern. A similar pattern was observed after incubation of PC with preformed lymphokines. The cyclic pattern was probably caused by a repeated action of lymphokines on spread macrophages, resulting in reversible and re-inducible inhibition.

Relation between reduced electrophoretic mobility of human lymphocytes and the inhibition of spreading of human monocytes caused by the same anti-human lymphocyte globulin.

Slowing of electrophoretic mobility of human peripheral blood lymphocytes after adding anti-human lymphocyte globulin (AHLG) in vitro correlated with indices of the monocyte spreading inhibition obtained by the same AHLG. Consequently, electrophoretic results may be associated with the prolongation of skin allograft survival in primates - the best and the only practical in vivo test for determination of the immunosuppressive potency of AHLG. If so, the simple electrophoretic test could be used in combination with other in vitro tests, preferably for monitoring of the AHLG production.

Immune reaction of tumor-bearing mice to Propionibacterium acnes and the antitumor effect of the bacteria.

The relationship between immunological reaction to Propionibacterium acnes (PA) and the antitumor effect of the injected bacterium was investigated. The aim was to determine whether the strength of the immune reaction to the bacterium can be used to predict its antitumor effectiveness. C3Hf/Sed mice received SC injections (right thigh) of viable cells of a methylcholanthrene-induced fibrosarcoma. When the tumor grew to 5 mm, the hosts received 350 micrograms PA IV as the antitumor treatment. Cellular immunity (footpad test) to PA was assayed in one group of these mice 14 days later, and in the other anti-PA agglutinins were determined 28 days later. The PA injection cured 22 of 58 mice in the first, and 20 of 46 mice in the second group. Footpad reaction and agglutinin titers to PA in cured mice were not statistically different from those in mice eventually killed by the tumor. Therefore, the strength of the immune reaction to PA in tumor-bearing mice could not be used to predict the antitumor effectiveness of the bacterium.

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.