Effect of graft versus host reaction on cell cycle time in neonatal mouse jejunum

Abstract. The duration of cell cycle parameters in control mouse jejunum has been compared with that found following induction of a graft-versus-host reaction (GvHR) during the first 3 weeks of postnatal life.
Values for t_c , and t_G1 were found to decrease progressively during normal development: estimates for the whole crypt column in 21-day-old mice were approximately half to one quarter those found 6 days after birth 12.1 ± 0.5 hr and 24.2 ± 0.3 hr for t_c ; 2.8 ± 0.3 hr and 12.1 ± 0.3 hr for t_G1 respectively; (means ± SE). t_S and t_G2 were found to remain approximately constant during this period of neonatal development.
Injecting foreign spleen cells into 3-day-old mice produced no effect on crypt cell proliferation or cell cycle parameters measured 3 days later. GvHR mice studied 8 days after spleen cell injection, however, showed both an increase in crypt cell proliferation and decreases in the values for t_c and t_G1 , to levels similar to those normally found in 21-day-old control animals ( t_c 12.4 ± 0.4 hr and t_G1 5.4 ± 0.4 hr for 11-day-old GvHR mice). The possible mechanism leading to these changes is discussed.
The ability of GvHR to stimulate cell proliferation is used in the present work to test the hypothesis that the total number of cell divisions taking place after birth determines the temporal sequence of changes in disaccharidase content produced during neonatal development.     

Parental spleen cells accelerate the development of intestinal brush border structure and function in neonatal mice

The influence of parental spleen cells on the postnatal development of brush border microvillus membrane structure and the ability to transport lysine and alanine has been studied in the mouse jejunum during the second week of postnatal life. Control tissue taken from 7-11 day old mice has an unchanging crypt-villus structure and a low enterocyte migration rate of about 1 micron hr-1. Microvillus elongation in crypt enterocytes takes 6 days to complete under these conditions. Lysine and alanine transport begin 2 days after structural differentiation has ceased. Parental spleen cells injected into 1-2-day-old F1 mice cause crypt cell hyperplasia, villus shortening and a 3-6-fold increase in enterocyte migration rate after a period of 8 days. These effects are associated with large reductions in the time needed to complete microvillus membrane development and first express absorptive function. Lysine and alanine transport begin approximately 6 hr after structural differentiation has ceased under these conditions. Adaptive changes in the development of enterocyte structure and function, induced by injection of parental spleen cells, bear some resemblance to other changes found to occur normally at weaning and in adult animals subjected to controlled changes in diet and environmental temperature. The possibility that common principles govern enterocyte adaptation and that some of these still apply in an intestine undergoing an immune reaction is discussed.     

Modulation of F1 cytotoxic potentials by GvHR. Host- and donor-derived cytotoxic lymphocytes arise in the unirradiated F1 host spleens under the condition of GvHR-associated immunosuppression

As an approach to dissect complex cellular events that lead to GvHR-associated immune disorders, we followed cytotoxic activities, including NK cytotoxicity, in the spleens of unirradiated F1 hosts undergoing GvHR induced by parental spleen cells. Spleen cells of (B10 X DBA/2)F1 or (B10 X AKR/J)F1 hosts undergoing GvHR induced by parental B10 spleen cells displayed a prompt and marked increase in NK cell activity within 36 hr, and the heightened activity lasted until day 8. The activity then declined abruptly and disappeared on day 12 of GvHR. Inversely, donor B10-derived CTL specifically directed to the opposite parental alloantigens of the F1 hosts emerged in these F1 host spleens on day 8, and the CTL activity reached a peak on day 12 when the host NK cell activity disappeared. During the period that the donor-derived anti-host CTL were present, these F1 host spleen cells lost not only NK cell activity but also the ability to mount in vitro CTL responses. In contrast, the respective F1 strain mice undergoing GvHR induced by the parental DBA/2 or AKR/J spleen cells showed only transient but marked increases in NK cell activity during the initial 36 hr, and then the activity decreased gradually to return to the normal level on day 10. In such GvHR F1 host spleens, donor-derived CTL could never be detected, and the spleen cells showed normal in vitro CTL responsiveness during the entire observation period of 16 days. These results are discussed from the viewpoint of genetically defined cellular events that lead to the GvHR-associated immune disorders.     

Cell population kinetics in pig epidermis: further studies

The cell population kinetics of the epidermis were studied in 4-month-old pigs. Mitotic figures were confined to the basal cell (L1) and the first suprabasal cell layer (L2). The mitotic index (MI) was 0.17 +/- 0.04% for L1 and 0.08 +/- 0.03% for L2. Labelled nuclei were distributed throughout the viable epidermis, the majority (79.1 +/- 1.1%) were in L1 with 19.5 +/- 1.2% in L2. The labelling indices (LI) in layers L1 and L2 were 7.1 +/- 0.4% and 3.4 +/- 0.1%, respectively. After labelling with two injections of tritiated thymidine [3H]TdR separated by 90 min, the LI increased to 8.2 +/- 0.3% in L1 and to 4.0 +/- 0.2% in L2. This increased labelling confirmed that cell proliferation occurs in both layers, L1 and L2, of the epidermis. The cell production rate (K) in L1 and L2 had an upper limit of 10.7 +/- 1.0 and 6.2 +/- 1.8 cells per 1000 cells per hour respectively. The cell flow rate per hour (cell flux), into and out of the DNA synthesis phase (S), and the duration of DNA synthesis were determined from double-labelling studies with [3H]TdR and [14C]TdR. The cell flux into and out of S was identical and was calculated as 0.6 +/- 0.1%/hr (L1) and 0.5 +/- 0.1%/hr (L2). Values for tS varied from 8 to 10 hr. The cell turnover times (tT) were in the range 89-129 hr and 180-261 hr for L1 and L2, respectively. Log normal curves were fitted to the fraction labelled mitoses data for L1 and L2. Values for tS for cells in L1 and L2 were 9.8 hr and 11.9 hr, respectively. tG2 + 1/2tM was 7.2 hr in L1 and 9.1 hr in L2.     

ALTERATION OF CELLULAR PROLIFERATION IN THE ILEAL EPITHELIUM OF SUCKLING AND WEANED RATS: THE EFFECTS OF ISOPROTERENOL

The purpose of this study was to analyse cell proliferation in the ileum before and after neonatal closure to macromolecules and to determine the effects of the sympathomimetic amine, isoproterenol (IPR), on cell cycle parameters. 8- and 28-day-old rats were employed in this study representing the neonatal periods of pre- and post-closure ileum respectively. The duration of the cell cycle phases was determined by the percentage of labeled metaphases technique (PLM) with computerized analysis of the curves. The generation cycle time was longer in 8-day-old suckling rats (18.63 hr) as compared to the older weaned rats (11.85 hr), and most of this 6.78 hr difference was in the G₁-period (4.5 hr). Other proliferative indices were also lower in the suckling rats—mitotic index (1.9 ± 0.4% as compared to 6.7 ± 0.9%), labelling index (26.8 ± 2.5% versus 44.2 ± 2.6%) and migration rate measured as per cent labeled villus cells (8.9 ± 3.1% versus 45.2 ± 3.4%). IPR was found to inhibit cellular proliferation in the ileal epithelium of both age groups. The cell cycle of the ileal epithelium of 8-day-old rats was lengthened from 18.63 to 21.07 hr and 28-day-old rats from 11.85 to 13.98 hr. IPR produced a decrease in mitotic index from 1.9 ± 0.4% to 1.6 ± 0.4% in pre-closure ileum and from 6.7 ± 0.9% to 5.1 ± 0.6% in post-closure ileum. Labeling index decreased from 26.8 ± 2.5% to 20.0 ± 2.0% in 8-day-old rats and from 44.2 ± 2.6% to 31.1 ± 3.0% in 28-day-old rats after IPR administration. There were also significant differences in growth fraction between age groups and a significant decrease in growth fraction after IPR-treatment. From the results of this study it appears that β-adrenergic stimulation has an inhibitory effect on neonatal ileal epithelium.     

Suppression of GvHR by in vitro allogeneically stimulated lymphocytes: the role of lymphoblasts in the regulation of a primary T-cell response

Graft versus host reaction (GvHR) induced in 10-day-old F1 mice by in vitro allogeneically or mitogenically stimulated spleen cells is lower than that induced by unstimulated fresh spleen cells. In vitro stimulated lymphoblasts are unable or only slightly able to induce a GvHR. An active suppression by the blasts is not involved. Since lymphoblasts after in vivo stimulation show an increased ability to elicit a GvHR it is concluded that in vitro and in vivo stimulated lymphoblasts have different properties. A different homing cannot be excluded after transfer to the mouse.     

Effects of accelerating growth on the onset of puberty in male mice

Sexual maturation was evaluated in male mice subjected to prenatal and preweaning overnutrition induced by reduction of litter size in embryonic and post-natal life. From birth to adulthood body weight was higher in overfed males than in controls. Plasma and testicular testosterone levels followed a similar pattern in normally fed and overfed males. Adult gonadotrophin levels were attained at 30 (FSH) and 40 (LH) days in controls and as soon as 20 days in overfed males. First fertile matings occurred at the same age in both groups. Overnourished males attained puberty when weighing considerably more than controls (34.7 +/- 0.4 vs. 29.4 +/- 0.3 g). The 30-day-old overfed males, which have attained the critical pubertal body weight of controls, were sexually immature. The data showed that in male mice puberty does not seem to be triggered by the attainment of a critical body weight.     

Immune dysfunction associated with graft-vs-host reaction in mice transplanted across minor histocompatibility barriers. II. Reversible defect in T-dependent antibody responses

B cell and Th cell functions were assessed in mice undergoing a graft-vs-host reaction (GvHR) in response to minor histocompatibility Ag by using the plaque-forming cell (PFC) response to the T-independent Ag TNP-Brucella abortus and the T-dependent Ag TNP-SRBC. Bone marrow plus spleen cells from B10.D2 mice were transplanted into lethally irradiated B10.D2 (syngeneic recipient) or H-2d-compatible BALB/c (allogeneic recipient) to produce a chronic form of GvHR. BALB/c recipients of an allogeneic transplant demonstrated a marked and proportional lymphoid depletion of the spleen with normal percentages of B cells, T cells, and CD4+ and CD8+ T cell subsets. Mice with GvHR made normal numbers of PFC/10(5) spleen cells in response to the T-independent Ag, but a significantly depressed number of PFC/10(5) spleen cells to the T-dependent Ag compared with normal B10.D2 mice and with irradiated B10.D2 recipients of syngeneic B10.D2 marrow plus spleen cells. Mice undergoing the minor Ag GvHR made significantly larger numbers of PFC/10(5) spleen cells after secondary immunization with TNP-SRBC compared with controls. In vitro assays demonstrated that B cells from mice with GvHR responded to T help from normal B10.D2 mice and that T cells from mice with GvHR provided help to normal B cells after in vivo immunization. These data demonstrate that radiation chimeras with GvHR in response to minor histocompatibility Ag have relatively normal B cell function and an apparent defect in T helper cell function that is reversible by immunization with appropriate Ag.     

Effects of Aflatoxin B1 Exposure and Sodium Selenite Supplementation on the Histology,Cell Proliferation,and Cell Cycle of Jejunum in Broilers

The aim of this study was to investigate the effects of aflatoxin B₁ (AFB₁) exposure and sodium selenite supplementation on the histology, cell proliferation and cell cycle of jejunum in broilers. A total of 240 1-day-old male AA broilers were divided into four groups of 60 each, fed with basal diet (control group), 0.3 mg/kg AFB₁ (AFB₁ group), 0.4 mg/kg supplement Se (Se group), and 0.3 mg/kg AFB₁?+?0.4 mg/kg supplement Se (AFB₁ + Se group) for 21 days, respectively. Compared with the control group, decreased jejunal villus height, villus height/crypt ratio, and proliferation cell nuclear antigen (PCNA)-positive cells, and G₂/M phase arrest and shedded epithelial cells on the tip of jejunal villus were observed in AFB₁ groups at 7 and 14 days of age. However, the villus/crypt ratio, PCNA-positive cells and cell percentage of G₀/G₁, S, and G₂/M phases had no significant differences between AFB₁ group and control group at 21 days. Simultaneous supplementation with sodium selenite restored these parameters to be close to those in control group. In conclusion, 0.3 mg/kg AFB₁ in the diet inhibits the development of broiler’s jejunum by reducing cellular proliferation and inducing G₂/M arrest during only the first 2 weeks after hatching. Supplementation of dietary sodium selenite at the concentration of 0.4 mg/kg Se had protective action against these toxic effects of AFB₁.     

The effect of continuous irradiation on cell proliferation and maturation in small intestinal epithelium.

Autoradiographic studies and scintillation counting of crypt material after pulse labelling with 3H-thymidine showed that during continuous irradiation with 290 rads/day a reduced proliferative activity is present in the crypts of rat small intestine after 1 day of irradiation and of normal activity during the remaining period (5 days) irradiation. After cessation of irradiation an increase in proliferative activity can be observed after 1 day of recovery. From the time (36-48 hr after starting of the irradiation) that the number of villus cells is reduced an expansion of the proliferation zone in the crypt was observed. Both effects last until 1 day of recovery after cessation of irradiation. The process of crypt cell maturation and of villus cell function has also been studied during and after continuous irradiation by micro-chemical enzyme analyses in isolated crypts and villi. It was found that the expansion of the proliferation zone in the crypt is accompanied by a decrease in activity of only those enzymes (i.e. non-specific esterases) which normally become active during crypt cell maturation. The activity of enzymes normally present mainly in the functional villus cells remained relatively unaffected by changes in crypt cell kinetics. A hypothesis of different regulation mechanisms of the proliferative activity in the intestinal crypt and a possible explanation of the different behaviour of various enzyme activities as a result of changes in crypt cell proliferation is discussed.     

Vitamin A prevents the irreversible proliferation of vaginal epithelium induced by neonatal injection of keratinocyte growth factor in mice

Exposure of female mice to estrogen during the perinatal period results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium when the animals become adults. However, the occurrence of such irreversible vaginal changes is blocked by concurrent vitamin A treatment. Neonatal exposure to keratinocyte growth factor (KGF), which is a paracrine mediator of epithelial-mesenchymal interactions, also induces the persistent proliferation and cornification of the vaginal epithelium in adult mice. This study was designed to examine whether concurrent administration of vitamin A inhibits the development of the irreversible vaginal changes in mice exposed neonatally to KGF. The vaginal epithelium in ovariectomized 35-day-old mice given 5 microg of KGF for 3 days after birth possessed a significantly larger number of layers and increased thickness as compared to that in control mice. Concurrent injections of 100 IU of vitamin A acetate inhibited the occurrence of the irreversible proliferation of the vaginal epithelium. These changes were equal to the results observed when 20 micro g of estrogen with or without vitamin A acetate was administered for 5 days after birth. Unlike the case of estrogen treatment, the effect of neonatal treatment with KGF seemed to appear after a latent period, since the vaginal epithelium did not show proliferation soon after the treatment. We discuss the inhibitory effect of VA on the irreversible vaginal changes induced by neonatal KGF treatment with reference to endocrine disruption by neonatal estrogen exposure.     

Testing the hypothesis that crypt size determines the rate of enterocyte development in neonatal mice

Pieces of mid-jejunum taken from 7-9 day old mice have been used to determine microvillus length in enterocytes located at different points along the crypt-villus axis to test the hypothesis that enterocyte development of structure is directly determined by the physical characteristics of the intestinal crypt. Parallel measurements of enterocyte migration rate were carried out using tritiated thymidine to determine the time course of microvillus elongation in neonatal mice. Microvillus length approximately doubled during early enterocyte migration from the crypt base to the lower part of the villus. Enterocyte migration rate was only 0.9 micron/hr at this stage of development, a value considerably less than that found in adult intestine. Results plotting the time dependency of microvillus elongation were fitted by a logistic curve giving a maximal rate for microvillus growth of 0.004 micron/hr. The corresponding estimate of crypt depth was 35 microns. Both these values are considerably less than those found in adult intestine. These results provide strong support for the general hypothesis that some factor associated with the physical length of the crypt, called crypt factor or CF, is directly responsible for controlling the way enterocytes organize subsequent structural differentiation of their surface membranes.     

Autoradiographic studies of glial proliferation in different areas of the brain of the 14-day-old rat

Cell cycle parameters, as well as the mode of proliferation of glial cells, in four different areas of the brain of the 14-day-old rat (cortex, corpus callosum, nucleus caudatus putamen and commissura anterior) were studied using different cell kinetic methods after injection of [3H]TdR and/or [14C]TdR. The duration of the S phase (tS) was found to be about 10 hr and that of the cycle time (tC) about 20 hr, tG2 is less than 2 hr and t(G2 + M) about 4 hr. These values are valid for glial cells in all four brain areas studied. However, the labelling index (LI) of the glial cells differs by a factor of 3, between 1.8 and 5.4% in the different brain areas. Accordingly, the growth fraction of the glial cell population in the four areas varies between 0.04 and 0.12. Glial cells (astrocytes as well as oligodendrocytes) proliferate according to a steady state system. Furthermore, the proliferation of glial cells is associated with continuous cell loss. After each mitosis about 3% of the daughter cells become pyknotic and die. In addition, a permanent exchange of glial cells occurs between the proliferating and non-proliferating pool.     

Neonatal immunity develops in a transgenic TCR transfer model and reveals a requirement for elevated cell input to achieve organ-specific responses

In recent years, it has become clear that neonatal exposure to Ag induces rather than ablates T cell immunity. Moreover, rechallenge with the Ag at adult age can trigger secondary responses that are distinct in the lymph node vs the spleen. The question addressed in this report is whether organ-specific secondary responses occur as a result of the diversity of the T cell repertoire or could they arise with homogeneous TCR-transgenic T cells. To test this premise, we used the OVA-specific DO11.10 TCR-transgenic T cells and established a neonatal T cell transfer system suitable for these investigations. In this system, neonatal T cells transferred from 1-day-old DO11.10/SCID mice into newborn (1-day-old) BALB/c mice migrate to the host's spleen and maintain stable frequency. The newborn BALB/c hosts were then given Ig-OVA, an Ig molecule carrying the OVA peptide, and challenged with the OVA peptide in CFA at the age of 7 wk; then their secondary responses were analyzed. The findings show that the lymph node T cells were deviated and produced IL-4 instead of IFN-gamma and the splenic T cells, although unable to proliferate or produce IFN-gamma, secreted a significant level of IL-2. Supply of exogenous IL-12 during Ag stimulation restores both proliferation and IFN-gamma production by the splenic T cells. This restorable form of splenic unresponsiveness referred to as IFN-gamma-dependent anergy required a transfer of a high number of neonatal DO11.10/SCID T cells to develop. Thus, the frequency of neonatal T cell precursors rather than repertoire diversity exerts control on the development of organ-specific neonatal immunity.     

Biophysical properties of hypoglossal neurons in vitro: intracellular studies in adult and neonatal rats

A brain stem slice preparation from adult and neonatal (less than or equal to 12 days old) rats and intracellular recordings were used to examine the cellular properties of neurons within the hypoglossal (HYP) nucleus. Resting membrane potential (Vm) for adult hypoglossal neurons was -80 +/- 2 (SE) mV. Rheobase was 2.1 +/- 0.4 nA, and input resistance (RN) was 20.8 +/- 1.5 M omega and decreased during the hyperpolarizing period ("sag"). Compared with adult HYP cells, newborn HYP neurons had significantly lower resting potentials (Vm = -73 +/- 2 mV), lower rheobase (0.7 +/- 0.2 nA), and higher RN (27.6 +/- 3.9 M omega). Single action potentials, elicited by short depolarizing-current pulses, were followed by a slow afterhyperpolarization in adult [6.4 +/- 0.3 mV, time constant (tc) 31.0 +/- 1.2 ms] and newborn cells (7.4 +/- 0.2 mV, tc 37.2 +/- 8.2 ms). Prolonged outward current (2 s) produced little spike frequency adaptation in either adult or newborn neurons. Onset of spike activity was not delayed by hyperpolarizing pulses preceding depolarizations. In addition, pharmacological experiments showed that HYP neurons have a tetrodotoxin-sensitive Na+ current and a delayed and an inward rectifier current but no major Ca2+ current. We conclude the following. 1) Electrophysiological membrane properties mature postnatally in HYP neurons; some of these developmental changes can be ascribed to an increase in soma size and dendritic outgrowth but others cannot. 2) Adult HYP neurons, compared with other brain stem neurons (i.e., vagal cells or cells in the nucleus tractus solitarius), are not endowed with major Ca2+ currents or K+ currents such as the A current and the Ca2(+)-activated K+ current.     

Suppressor T cells derived from early postnatal murine spleen inhibit cytotoxic T-cell responses

Postnatal T-suppressor cells have been detected in a number of experimental systems. They have been shown to inhibit humoral responses, proliferation in a mixed-lymphocyte reaction and the induction of killer cells. The suppressor function observed in the postnatal mouse does not appear to be antigen specific and its ontogeny may be influenced by other cell types and by serum factors such as α-fetoprotein. We have detected a nonadherent, radioresistant splenic T cell present in neonatal mice ranging in age from 1 to 9 days which can nonspecifically suppress killer cell induction. This suppressor cell must be cultured in vitro in order to function, but it does not require alloantigen to be induced. Adult spleen cells tested in the same system yield antigen-specific T-cell suppression. Our results suggest that the nonspecific suppressor detectable in 1- to 9-day-old mice disappears in adult life, and is replaced by antigen-specific suppressors. The biological role of these suppressors is discussed.     

Development of body oxygen stores in harbor seals: effects of age, mass, and body composition

Harbor seal pups are highly precocial and can swim and dive at birth. Such behavioral maturity suggests that they may be born with mature body oxygen stores or that stores develop quickly during the nursing period. To test this hypothesis, we compared the blood and muscle oxygen stores of harbor seal pups, yearlings, and adults. We found that pups had smaller oxygen stores than adults (neonates 57%, weaned pups 75%, and yearlings 90% those of adults), largely because neonatal myoglobin concentrations were low (1.6+/-0.2 g% vs. 3.8+/-0.3 g% for adults) and changed little during the nursing period. In contrast, blood oxygen stores were relatively mature, with nursing pups having hematocrit (55%+/-0.2%), hemoglobin (21.7+/-0.4 g%), and blood volume (12.3+/-0.5 mL/kg) only slightly lower than the corresponding values for adults (57%+/-0.2%, 23.8+/-0.3 g %, and 15.0+/-0.5 mL/kg). Because neonatal pups had relatively high metabolic rates (11.0 mL O2/kg min), their calculated aerobic dive limit was less than 50% that of adults. These results suggest that harbor seals' early aquatic activity is primarily supported by rapid development of blood, with immature muscle oxygen stores and elevated use rates limiting aerobic diving ability.     

Age-dependent lipid peroxidation in neonatal rat lung tissue.

A unique, age-specific pulmonary lipid peroxidation has been found to occur after incubation of neonatal rat lung homogenates in the absence of any added factors. As measured by the formation of malondialdehyde, lipid peroxidation was not detectable in rat lung homogenates prepared from animals immediately after birth but appeared by the second day and reached a maximum at 5 days of age. The effect gradually disappeared by 20 to 21 days after birth. The addition of NADPH did not enhance lipid peroxidation in the sensitive age group nor did it initiate lipid peroxidation when added to lung homogenates from either 1-day-old or adult rats. The activities and concentrations of various endogenous antioxidants were measured in neonatal lung tissue. When measured in lung tissue obtained from rats during the sensitive age period, no concomitant deficiencies of glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase, reduced glutathione, or a-tocopherol were observed. With the exception of α-tocopherol, none of these factors inhibited malondialdehyde formation when added to homogenized lung tissue prepared from 5-day-old rats. α-Tocopherol did inhibit malondialdehyde formation in 5-day-old rat lung homogenates but at a concentration much greater than the endogenous concentration found in adult rat lungs. The 21-day neonatal age period during which malondialdehyde is produced following incubation of lung tissue is similar to the 3-week period immediately after birth reported to be the time of maximum proliferation of rat lung fibroblasts, type 1 pneumocytes and type II pneumocytes.     

Monoclonal antibody 4H12 recognizes subsets of adherent-lymphokine activated killer cells and splenic natural killer cells from pregnant and neonatal mice

Using a new mAb, 4H12, that recognizes a plasma membrane-associated Ag on granulated metrial gland cells, we identified subsets of murine NK cells in spleen cell-derived adherent lymphokine activated killer cells and in the spleens of neonatal and pregnant mice. In spleen cell adherent-lymphokine-activated killer cultures, 4H12 Ag was detected on a small subset of cells after 7 days culture and expression increased with time to 70% of the cells after 21 days culture. 4H12+ cells were large (up to 70 microns) and granular. The Ag was also detected in the cytoplasmic granules of some, but not all 4H12 surface positive cells. Coexpression studies indicated 4H12+ cells were predominantly positive for the NK1.1 and ASGM1 Ag, negative for the MAC-1 and F4/80 Ag, and +/- for the LGL-1 and CD3 Ag. Subsets of 4H12+/-, LGL-1+/- exhibited morphologic characteristics restricted to specific phenotypic subsets. The 4H12-/LGL-1+ subset was shown to contain the smallest, least granular cells, whereas the 4H12+/LGL-1+/- subsets contained the largest and most granular cells. Although 4H12 expression was negligible in the spleens of normal adult mice, spleen cells of neonatal and pregnant mice contained subsets of NK1.1+ cells that coexpressed 4H12. The 4H12+/NK1.1+ and 4H12-/NK1.1+ subsets displayed differential levels of NK1.1 expression. 4H12+ NK cells were NK1.1 low to high, whereas 4H12- NK cells were NK1.1 high only. The functional significance of subsets of NK cells in IL-2 culture and in the spleens of neonatal and pregnant mice remains to be elucidated.     

THE EFFECT OF CONTINUOUS IRRADIATION ON CELL PROLIFERATION AND MATURATION IN SMALL INTESTINAL EPITHELIUM

Autoradiographic studies and scintillation counting of crypt material after pulse labelling with ³H-thymidine showed that during continuous irradiation with 290 rads/day a reduced proliferative activity is present in the crypts of rat small intestine after 1 day of irradiation and of normal activity during the remaining period (5 days) irradiation. After cessation of irradiation an increase in proliferative activity can be observed after 1 day of recovery. From the time (36–48 hr after starting of the irradiation) that the number of villus cells is reduced an expansion of the proliferation zone in the crypt was observed. Both effects last until 1 day of recovery after cessation of irradiation. The process of crypt cell maturation and of villus cell function has also been studied during and after continuous irradiation by micro-chemical enzyme analyses in isolated crypts and villi. It was found that the expansion of the proliferation zone in the crypt is accompanied by a decrease in activity of only those enzymes (i.e. non-specific esterases) which normally become active during crypt cell maturation. The activity of enzymes normally present mainly in the functional villus cells remained relatively unaffected by changes in crypt cell kinetics. A hypothesis of different regulation mechanisms of the proliferative activity in the intestinal crypt and a possible explanation of the different behaviour of various enzyme activities as a result of changes in crypt cell proliferation is discussed.