Consequences of neonatal thymectomy in New Zealand black mice

The possible role of the thymus in autoimmune disease was studied by comparing the effects of neonatal thymectomy on New Zealand Black (NZB) mice (which develop a Coombs positive haemolytic anaemia) and on C3H/Bi, F₁ (C57BL × C3H/Bi), C57BL and TO mice.

The neonatally thymectomized NZB mice, in common with those of other strains, showed lethal wasting, a lymphocyte deficit in their lymphoid organs and blood, their packed cell volume was reduced and some had liver lesions associated with the hepatotrophic virus MHV-1. As in C3H/Bi and hybrid mice, thymectomy had little effect on the levels of immunoglobulins (IgG, IgA, IgM) present in their sera.

Removing the thymus from NZB mice did not prevent and could precipitate Coombs positive reactions; thymectomized mice of the other strains were Coombs negative. Although germinal centres develop and plasma cells occur in the lymphoid organs of most thymectomized mice, the striking feature of the NZB mice was the large number, size and activity of the germinal centres that persisted after thymectomy.

     

In vivo Bactericidal Activity of Mouse Complement Against Esch. coli

Live Escherichia coli of complement sensitive and resistant strains were labelled with ¹⁴C and injected i.v. into normal mice and into a co-isogenic strain deficient in C′5. The fate of the bacteria was followed by determining total and viable counts in blood samples taken at intervals over a 30 min. period and in homogenates of the liver, spleen, lungs and kidneys taken at the end of the experiment.The results show that sensitive bacteria can be killed by mouse complement within the circulation and suggest that complement may also play a part in the intracellular killing of Esch. coli in some organs.     

Studies on thymus products: III. Epithelial origin of the serum thymic factor

Serum thymic factor (TF) has been tested by its action on spleen rosette-forming cells from adult thymectomized mice. It has been confirmed in a blind study using coded serum samples that TF disappeared early after adult thymectomy and reappeared after grafting a thymus, either as a free graft or enclosed in cell impermeable diffusion chambers. Similar reconstitution was also obtained by grafting a non-lymphoid epithelial thymoma or pure epithelial thymus, obtained by in vivo incubation of a thymus within a diffusion chamber in an intermediate host. Conversely, TF levels were not restored in thymectomized animals treated with dispersed spleen cells or with dispersed thymic lymphocytes.     

Effect of neonatal thymectomy on experimental autoimmune hepatitis in mice.

Using an autoimmune hepatitis model of A/J mice which was prepared with immunization by syngeneic crude liver proteins, various influences of neonatal thymectomy were studied by observations of histological liver changes, autoantibody to liver-specific membrane lipoprotein (LSP), delayed-type hypersensitivity (DTH) to LSP, and purified protein derivative (PPD), and suppressor activity to LSP. The liver changes in the thymectomized mice were more intense than those in the non-thymectomized controls. Production of the anti-LSP autoantibodies and positive DTH to syngeneic LSP could be recognized in both groups of the thymectomized mice and the non-thymectomized controls, but the levels of those were higher in the former. In the level of DTH to PPD the thymectomized mice were lower than the non-thymectomized controls. Adoptive transfer experiments showed that suppressor activity to LSP was reduced in the spleen cells of neonatally thymectomized mice. This experiment suggests that neonatal thymectomy is apt to abolish tolerance to LSP on account of depressed suppressor activity to autoantigen, and accordingly liver damage is increased.     

Changes produced by pertussis antigen on the blood cells and lympho-haemapoietic tissues after early and late thymectomy or splenectomy

The effect of thymectomy and splenectomy in C3H/Bi mice on the responses of circulating leucocytes and on morphological changes of the haematopoietic tissues after injection of pertussis vaccine has been studied.

After pertussis all mice showed depletion of lymphoid cells in all the lymphoid organs as well as in bone-marrow and an increased number of leucocytes, lymphocytes, neutrophils and monocytes in the circulation. Neonatal thymectomy decreased lymphocytosis produced by pertussis. Thymectomy, at all ages studied, fostered an increase in the number of monocytes and polymorphonuclears in circulation. Splenectomy at birth or early in life provoked an increase in levels of circulating polymorphonuclears and lymphocytes in pertussis treated animals.

In neonatally thymectomized mice the depletion of lymphoid cells from lymphoid tissues after pertussis could be shown to include the thymic-independent areas. The depletion of small lymphocytes from thymus following pertussis persisted longer than depletion of small lymphocytes from spleen, marrow or lymph nodes. The longer persistence of lymphoid depletion in the thymus than in peripheral lymphoid tissues is, we believe, to be related to the central lymphoid function of thymus as a site of differentiation of lymphoid cells and to the aloofness of thymus from recirculation of fully differentiated peripheral lymphocytes.

     

Effects of Arvin in mice. Immunofluorescent and histochemical studies

The in vivo response to injection of a purified fraction of Malayan pit viper venom, Arvin, has been studied in mice. Fibrin “microclots” were localized by means of immunofluorescence mainly in the lungs and to a lesser extent in the kidneys, liver and spleen. Increased fibrinolysis in these organs was demonstrated by means of the histochemical slide method. Inhibition of fibrinolysis in the tissues by soy bean trypsin inhibitor and epsilon-aminocaproic acid prior to Arvin injection was followed by significantly increased deposition of fibrinogen and/or its derivatives with formation of thrombi and death of 50% of the animals. These observations indicate the importance of tissue activation of plasminogen and the rapid lysis of microclots in clinical defibrination by Arvin.     

Effect of early thymectomy on the cellular changes occurring in the spleen of the clawed toad following administration of soluble antigen

Xenopus toadlets thymectomized as larvae at 8 days post-fertilization developed relatively normal lymphoid tissues but the spleen was reduced in weight. The increase in numbers of pyroninophilic cells and in the size of the white pulp regions which occurs in the spleen of intact, control Xenopus in response to administration of human γ-globulin (HGG) in adjuvant was less marked in thymectomized animals.

HGG localization in an outer zone of the white pulp was demonstrated by immunofluorescence in the spleens of intact Xenopus. Following early thymectomy this reaction was abrogated or severely diminished, as was also the production of circulating antibody to HGG.

     

Studies on acquired immunity to coccidiosis in bursaless and thymectomized fowls

Fowls hatched from embryos inoculated in ovo with testosterone between the sixth and ninth day of incubation hatched without a detectable bursa of Fabricius. These fowls failed to develop antibodies as the result of repeated infection with Eimeria tenella and levels of serum immune globulin were usually markedly reduced or undetectable. There were very few pyroninophilic cells in the caeca or spleen and secondary foci in the spleen and caecal lymphoid tissue were either not detected or were very reduced in number. The spleen and thymus weights were significantly reduced by testosterone treatment.

Nevertheless, these fowls were successfully immunized, so that they resisted infection when challenged with viable oocysts of E. tenella.

Active E. tenella infection of normal, susceptible, control fowls significantly reduced the thymus weight and increased the bursa weight. Severe haemorrhage into the caecal lumen of infected fowls resulted in lowered blood erythrocyte and lymphocyte counts and a reduction in the total serum protein.

Antibodies, capable of lysing sporozoites, were detected in normal fowls after immunization and in normal susceptible fowls 5 days after initial infection.

Complete surgical thymectomy was attempted within the first 1½ hours after hatching. However, this was only exceptionally complete and about 10 per cent of thymic tissue was detectable at subsequent post-mortem 56 days later. Thymectomized birds produced antibody, pyroninophilic cells and secondary spleen foci indistinguishable from normal control immunized chickens. However, there was a significant reduction in the number of small lymphocytes in the blood. Although thymectomized fowls were successfully immunized against E. tenella infection, there was an indication, shown by daily oocyst discharge determinations that thymectomized fowls were less resistant during immunization than normal fowls. However, both groups of fowls, when challenged, were fully immune. The significance of partial thymectomy and the time of thymectomy in the fowl in relation to the acquisition of resistance is discussed.

The results support our previous observations which have failed to demonstrate a significant role for humoral antibody in the mediation of resistance to E. tenella.

     

Size of the population of CD4+ natural killer T cells in the liver is maintained without supply by the thymus during adult life

Given that there are few natural killer T (NKT) cells in the liver of athymic nude mice and in neonatally thymectomized mice, it is still controversial whether all NKT cells existing in the liver are supplied by the thymus or if some such cells develop in the liver. To determine whether or not NKT cells are consistently supplied from the thymus during adult life, thymectomy was conducted in mice at the age of 8 weeks. Interestingly, the proportion and number of CD4+ NKT cells increased or remained unchanged in the liver after adult thymectomy and this phenomenon continued for up to 6 months after thymectomy. The administration of alpha-galactosylceramide induced severe cytopenia (due to apoptosis) of CD4+ NKT cells in the liver on day 1, but subsequent expansion of these NKT cells occurred in thymectomized mice similar to the case in normal mice. However, in thymectomized mice given lethal irradiation (9.5 Gy) and subsequent bone marrow transfer, the population of CD4+ NKT cells no longer expanded in the liver, although that of CD8+ NKT cells did. These results suggest that thymic CD4+ NKT cells, or their progenitors, may migrate to the liver at a neonatal stage but are not supplied from the thymus in the adult stage under usual conditions. CD8+ NKT cells can be generated in the liver.     

TISSUE REACTIONS TO ASPERGILLUS IN CASES OF HODGKIN'S DISEASE AND LEUKAEMIA

Five cases of aspergillosis complicating Hodgkin's disease and leukaemia are reported. The organs involved were: lungs (all five cases), stomach (Case 3); brain and meninges (Case 4); heart, kidneys, spleen, thyroid, and liver (Case 2). Cultures of Aspergillus fumigatus were obtained from the post-mortem tissues of three patients.     

Pharmacokinetics of an immunomodulator peptidoglycan monomer in mice after intravenous administration

A ¹⁴C labeled low molecular weight immunomodulator, peptidoglycan monomer (¹⁴C-PGM), was injected intravenously (i.v.) into mice. At various time intervals thereafter (15 min – 6 h), radioactivity in the urine, whole blood, plasma, kidneys, liver, spleen, lungs, intestines and the brain of the mice was determined. Shortly after injection, ¹⁴C-PGM was very rapidly excreted from the organism, so that 1 h following administration, 80% of the radioactivity was found in the urine (62% as unchanged PGM and the rest as the metabolites pentapeptide and disaccharide). At the same time, around 2% of the injected material was found in the blood. Six hours after injection, equal quantities were found in the intestines, liver and blood (0.5%), slightly less in the kidneys, lungs and spleen (0.2–0.3%) and the least quantity in the brain (0.04%). However, the dynamics of retention in the organs was evidently different. In the kidneys, lungs and spleen, radioactivity steadily decreased over the studied period. In the liver following an initial decrease, radioactivity remained the same 3 and 6 h after injection. On the other hand, in the intestines and brain PGM seemed to accumulate rather than disapper following i.v. administration. This fact should be considered when explaining different biological activities of low molecular weight bacterial peptidoglycans.     

Tissue stages of<Emphasis Type="Italic"> Hepatozoon canis</Emphasis> in naturally infected dogs from São Paulo State,Brazil

A total of 222 dogs were examined by blood smear examination and Hepatozoon canis infection was detected in 13 dogs (5.9%). Five H. canis-infected dogs were necropsied to observe tissue stages in the organs. Fragments of spleen, liver, lungs, heart, kidneys, lymph nodes, bone marrow and skeletal muscles were used to made touch-impression smears. No macroscopic lesions were found in the organs. Two dogs had gamonts within polymorphonuclear cells and schizonts in various stages of development within the spleen and the bone marrow. Nevertheless, no mature meronts were found.     

Distribution of thietazole in organs and tissues of rats after single and repeated administration

We studied the kinetics of thietazole distribution in the liver, brain, kidneys, spleen, heart, skeletal muscles, lungs, adipose tissue, and testicles after single and repeated administration of this drug. Single and repeated administration of thietazole was followed by elimination of this drug from the blood into organs and tissues. After repeated administration, thietazole was selectively accumulated in the spleen. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 555–557, May, 2008     

Distribution of ethomerzol in organ and tissue of rats after single and course treatment

We studied experimental kinetics of ethomerzol (5-ethoxy-2ethylthiobenzimidazole hydrochloride) distribution in the liver, brain, kidneys, spleen, heart, skeletal muscles, lungs, adipose tissue, and testes of rats after its single or course administration. Peculiarities of ethomerzol distribution in various administration regimens were analyzed. Single treatment led to more pronounced accumulation of the drug in the liver. Study of ethomerzol distribution after course treatment revealed organs and tissues accumulating the drug (blood, brain, heart, kidneys, lungs, and adipose tissue). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 1, pp. 47–49, January, 2008     

Induction of autoimmune hepatitis and autoantibodies to liver antigens by neonatal thymectomy in mice

We examined development of autoimmune hepatitis in neonatally thymectomized C3H/HeN mice and tried to characterize the nature of liver antigens recognized by the autoantibodies at the molecular level. Autoantibodies to crude liver proteins detected by ELISA were found in 12 (67%) of 18 mice thymectomized 2 days after birth. However, autoantibodies were not detected in mice thymectomized 7 days after birth. The autoantibodies mainly consisted of IgG and reached the maximum level 8 weeks after birth. Hepatic inflammation, mononuclear cell infiltration in the portal area, was seen in 5 (28%) of 18 mice thymectomized 2 days after birth, but not in mice thymectomized 7 days after birth. Most infiltrating cells were Thy-1⁺ lymphocytes. The serum autoantibody level to crude liver proteins in mice with hepatitis was much higher than that in mice without hepatitis. We fractionated crude liver proteins by a Sepharose 6B column and examined the reactivity against the autoantibodies. The autoantibodies of three of five mice with hepatitis reacted with the ≈150 kD liver proteins other than liver-specific protein (LSP). By Western immunoblotting of SDS–PAGE using LSP and fractionated liver proteins, we found that the molecular weights of the target antigens were 52 kD in LSP and 150 kD (strong band), 138, 128, 120 and 110 kD (weak band) in fractionated liver proteins other than LSP. This 150-kD target molecule in crude liver proteins was found only in liver. These results indicate that hepatitis and autoantibodies to liver proteins are induced spontaneously by neonatal thymectomy in mice, and the candidates of autoantigen in this hepatitis model are 52-kD protein in LSP and 150-kD liver proteins different from LSP. Still more, we regard the 150-kD molecule as a new autoantigen related to hepatitis.     

Recovery of immunological responsiveness in thymectomized mice

After a limited period of immunological unresponsiveness, neonatally thymectomized colony-bred Swiss mice were found to recover their ability to form haemagglutinins and haemolysins as well as their antibody-plaque-forming capacity following injection of sheep erythrocytes. No such spontaneous reconstitution was observed in F₁-hybrids of highly inbred CBA and CBA-T6T6 mice. Adult thymectomized and irradiated Swiss mice similarly regained their ability to form haemolysins and haemagglutinins, but no regeneration of antibody-plaque production occurred in these mice during the period of observation. No regular correlation was found between the degree of immunological deficiency on the one hand and the level of circulating lymphocytes or the histological appearance of the spleens on the other, following neonatal thymectomy or adult thymectomy and irradiation.

The possible mechanism of recovery from immunological impairment after thymectomy and the apparent discrepancies between overall haemolysin production and haemolytic plaque production in the spleen are discussed.

     

Quantitative studies of lymphocytes and other cell populations in the bone marrow of neonatally thymectomized C3H mice

The effects of neonatal thymectomy on the development of the lymphoid, erythroid and granulocytic cell populations in mouse bone marrow have been assessed by quantitative techniques. The numbers per unit volume of bone marrow of 17 cell types were determined in neonatally thymectomized and sham thymectomized C3H mice at two, four and eight weeks of age, and compared with those of normal C3H mice. After neonatal thymectomy the numbers of small lymphocytes, large and medium-sized lymphoid cells, and erythroid cells reached normal levels at two weeks but fell progressively to 18%, 22% and 42% of normal, respectively, by eight weeks. In sham thymectomized mice these cell populations did not differ significantly from normal. Immature and mature granulocytes were elevated in numbers two weeks after either neonatal thymectomy or sham thymectomy, suggesting a transient non-specific stimulation of granulocytopoiesis. During continuous infusion of ³H-thymidine for ten days in neonatally thymectomized mice aged four weeks and eight weeks many bone marrow small lymphocytes remained unlabeled. The results demonstrate that early postnatal development of bone marrow lymphoid and erythroid cells proceeds normally in the absence of the thymus, in accord with the concept of the bone marrow as a primary site of lymphocyte production and differentiation. In addition, some slowly-renewing small lymphocytes in bone marrow appear to be thymus-independent cells.     

Immunobiology of Germfree Mice Infected with Nocardia asteroides

Nocardia asteroides GUH-2 was administered either intranasally or by intravenous inoculation into (i) conventionally grown, (ii) germfree, and (iii) lipopolysaccharide-treated germfree NIH:S mice. The number of bacteria within the lungs, brain, kidneys, adrenals, liver, spleen, and blood was quantitated at 3, 24, 72, and 168 h after infection. Further, the histological changes that occurred in each of these organs after infection were studied. The data demonstrated that germfree mice were significantly more susceptible to the acute phase of infection caused by N. asteroides than the conventionally grown animals. The brains and lungs of these mice were affected most dramatically. Pretreatment of the germfree mice with lipopolysaccharide completely reversed this enhanced susceptibility and rendered the animals more resistant to infection than the conventionally grown mice. These observations establish further the role of macrophage activation and the development of cell-mediated immunity in host resistance to nocardia. In addition, the presence of a resident microflora within the host appears to be important in the development of resistance to systemic nocardial infections.     

The immunological responsiveness of germ-free mice thymectomized at birth. II. Lymphoid tissue and histopathology

The effect of neonatal thymectomy and antigenic stimulation on the lymphoid cell population has been studied in germ-free mice. Neither thymectomy nor injection of sheep erythrocytes induced any significant alteration in the blood lymphocyte levels. There was a clear-cut reduction in the cellularity of the periarteriolar lymphocyte sheaths of the spleen and of the paracortical regions of the lymph nodes in the thymectomized mice. Following stimulation with sheep erythrocytes, large pyroninophilic cells appeared in these areas in the intact germ-free controls but in only a few thymectomized mice and then in reduced numbers. Thymectomy did not influence the cellularity of the lymphoid follicles but less germinal centre and plasma cell activity occurred in response to an injection of sheep erythrocytes. Lesions suggestive of autoimmune reactivity were not found in lymphoid or nonlymphoid tissues of neonatally thymectomized germ-free mice. Lesions typical of viral infections were seen in some germ-free mice in both thymectomized and intact groups. It is concluded that the specific defect associated with the absence of the thymus is a reduction in a particular class of lymphocytes the development of which is under thymus control and the activities of which are to mediate certain defined immunological responses.     

Malignant changes in New Zealand black mice

Ageing, Coombs positive, NZB mice, may spontaneously develop a neoplasia of the reticulum cell type which can be transferred by serial passage of their lymphoid tissues in young intact or neonatally thymectomized syngeneic recipients. The recipients (103/117) of cell suspensions prepared from the enlarged spleens and/or lymph nodes of four such donors developed an extensive and lethal reticulum cell neoplasia affecting the spleen, lymph nodes, lungs and liver but the bone marrow, thymus and kidneys were seldom involved. The recipients (16/17) of spleen cells from a fifth donor showed massive proliferations of eosinophils in all the organs examined.

Prematurely positive antiglobulin (Coombs) reactions were detected in only two recipients. Although there was an indication that the IgM content of the sera decreased as one of the passages progressed, the levels of IgG and IgA were not seriously distorted.

Particles resembling murine leukaemia virus were identified by electron microscopy in the spleen and in plasma or serum pellets of passage recipients. However, similar particles were also seen in the thymus and/or spleen or bone marrow of untreated NZB mice including an 18-day embryo and animals aged 1–56 weeks, although no particles were found in plasma and serum pellets of mice aged 6–70 weeks.

The theory that autoimmunity, malignancy and virus infection are directly related is discussed.