Monitoring silver diamine fluoride application with optical coherence tomography.

The objective of this study was to investigate the use of optical coherence tomography (OCT) for monitoring changes in the structure of caries lesions overtime after treatment with silver diamine fluoride (SDF). Artificial caries lesions were formed on dentin bovine blocks. Each block was partitioned into 5 windows: one lesion was covered by nail varnish as control (LC), one sound window was covered with nail varnish (SC), one sound window was exposed to SDF (SCT), one lesion received 2 applications of SDF (L2), while the other lesion received one application of SDF (L1). Each window was scanned using OCT before SDF application, and every week subsequently, for 12 weeks after initial SDF treatment. Parameters such as mean intensity and the width of the peak of increased reflectivity located at the sample surface and the intensity at a depth of 180-µm were monitored. High-resolution microscopy was also used to for the analysis of selected samples. Changes in the parameters measured showed significant changes on dentin lesions after SDF application. OCT resolved structural changes after SDF application as well as changes overtime. High resolution microscopy images confirm penetration of SDF into the samples. Such changes can potentially be monitored to determine if and when re-application of SDF is needed.

Thermal Imaging of Root Caries In Vivo.

Improved methods are needed to assess the structure and activity of lesions on root surfaces in order to improve clinical decision making. Conventional visual and tactile methods for assessing lesion activity are not reliable, and the clinician is often unable to evaluate if the lesion is progressing or has remineralized. An important marker of an arrested lesion is a highly mineralized surface zone that forms when mineral is deposited in the outer layer of the lesion. In vitro studies have shown that a mineralized surface zone influences the kinetics of water evaporation and the surface temperature while drying. Temperature changes can be monitored by measuring the thermal emission with thermal imaging. Studies have also shown that the depth and severity of demineralization and the thickness of the highly mineralized transparent surface zone on arrested lesions can be measured nondestructively with optical coherence tomography (OCT). Thermal imaging at 8-µm to 13-µm wavelengths was completed on 30 test subjects with a suspected active root caries lesion by monitoring thermal emission from the tooth surfaces during 30 s of air drying. Lesions were also evaluated using cross-polarization OCT (CP-OCT) during lesion dehydration to identify transparent surface zones indicative of arrested lesions and determine if shrinkage occurred during drying. The overall thermal emission recorded during drying was significantly different (P < 0.001) when comparing sound tooth surfaces, lesion areas identified as arrested, and lesion areas identified as active, demonstrating that thermal imaging is a promising approach for the clinical assessment of lesion activity on root surfaces. Ten of the lesions in this study had distinct areas with transparent surface zones that were visible in CP-OCT images. Shrinkage was detected with CP-OCT during drying for 12 lesions. This study confirms that these novel approaches for assessing lesion activity on root surfaces can be implemented in vivo.

Vertical distraction of fibula transplant in a case of mandibular defect caused by shotgun injury.

This case demonstrates the successful aesthetic and functional reconstruction of a complex facial gun-shot injury with extended bone defects and soft tissue destructions using a 3-step procedure. Initially, a reconstruction plate was inserted, later a fibula transplant enabled the basic reconstruction and finally was distructed in a 3rd session. The rationale behind the sequencing of surgical sessions was the extended bony defect and soft-tissue destruction. The main problem in this type of wound is hypoxia or anoxia of the receptor bed for the transplant. A microvascular anastomosized bone transplant is necessary for sufficient oxygen tension in the recipient site. The anatomical dimensional disproportion of the transplanted free fibula graft and the shape of the mandible were corrected prior to the insertion of dental implants by means of vertical distraction.

Effects of cyclophosphamide and of busulfan on spleen colony-forming units and on hematopoietic stroma.

The effects of cyclophosphamide (CY) and busulfan (BU) on the hematopoietic stromal function (HS-P) of mouse marrow were evaluated. Stromal function of femoral marrow was assessed by implanting the test femur s.c. into an isogeneic host and determining the number of CFU-S in the implant 6 weeks later. Since the CFU-S have been shown previously to be primarily of host origin, this presumably measures the abilit- of donor hematopoietic sites to harbor host CFU-S. After injection of CY, the number of CFU-S in the marrow fell but recovered to normal within 6 weeks. The HS-P function fell to half-normal after 10 mg of CY and did not regenerate detectably in 6 weeks. On the other hand, 2 mg of BU i.p. caused a lesser initial decline in the number of CFU-S, but recovery was still incomplete after 6 weeks. BU given p.o. had a more marked effect on CFU-S and caused a significant decline in the HS-P function. Doses of CY (5 mg/dose) given intermittently appear to cause cumulative damage to HS-P function. HS-P function did not, in any experiment, recover significantly in the 6 weeks following the last dose of CY. This result suggests that large doses of the alkylating agent CY causes prolonged and perhaps permanent HS-P damage. This damage to the HS-P is cumulative when the CY is given at weekly intervals. Despite lack of HS-P recovery, CFU-S regenerate rapidly after CY therapy is stopped. On the other hand, BU also causes damage to the HS-P. However, even when BU is given at a dosage that does not significantly affect the HS-P, CFU-S recovery is delayed, suggesting that BU affects the CFU-S in a manner that differs qualitatively from that of CY.

Induction of lymphomas in mice by busulfan and chloramphenicol.

Busulfan causes long-lasting defects in the hematopoietic stem cells and in the immune system of mice. We designed studies to determine whether chloramphenicol further damaged the already defective hematopoietic stem cells of mice that were pretreated with busulfan, and we unexpectedly observed that mice given injections of the combination of busulfan and chloramphenicol developed lymphomas in relatively high incidence. The disease is invariably associated with splenomegaly and enlargement of the thymus. Leukocytosis with lymphoblasts in the peripheral blood occurred in some affected mice. The malignant cell is a lymphoblast of thymic origin. Thirteen of 37 mice which received both busulfan and chloramphenicol developed lymphomas. An additional five of the remaining 24 mice without proven lymphoma died and were not autopsied. Twelve of the 13 proven lymphomas developed within 280 days from the start of the experiment. Four of the 35 mice treated with busulfan alone developed lymphomas, and an additional five of the remaining 31 died but were not autopsied. Two of 41 mice treated with only chloramphenicol developed lymphomas. Of the mice treated with either busulfan or chloramphenicol alone that developed lymphomas, all did so more than 280 days from the start of the experiment. None of the control mice developed lymphoma. We conclude that both busulfan and chloramphenicol may induced lymphomas in mice that are not known to develop them spontaneously. The combination of both busulfan and chloramphenicol increased the frequency and accelerated the onset of the disease.

Dynamics of leukemic and normal stem cells in leukemic RFM mice.

RFM mice spontaneously develop a myelogenous leukemia that is transplantable into nonleukemic RFM mice. On transplantation, hemopoietic stem cells from leukemic mice (L-CFU-S) will seed in the spleen and grow as discrete colonies, as will hemopoietic stem cells from normal mice (N-CFU-S). As the leukemic cells used in these experiments have 39 chromosomes and normal murine cells have 40, it has been possible to estimate the numbers of N-CFU-S and L-CFU-S in RFM mice at weekly intervals after these mice had been given i.v. injections of 10(6) leukemic spleen cells (spleen cells from preterminal leukemic mice). At each study time, splenic weights, peripheral blood counts, and nucleated cell counts and colony forming units (CFU-S) of marrow, spleen, and blood were assayed. The karyotypes of dividing cells from and the histology of the resultant spleen colonies were also studied. Two weeks after the injection of leukemic spleen cells, the number of CFU-S in the marrow had increased to 3 to 10 times normal, that in the spleen to 100 times normal, and that in the blood was markedly increased. Three weeks after injection, the number of CFU-S in the marrow fell from the peak level at 2 weeks, the number in the spleen rose modestly, and the number in the blood continued to be markedly increased. A normal distribution of erythroid, myeloid, and megakaryocytic colonies was obtained from CFU-S assayed 1 week after injection of leukemic spleen cells, but from CFU-S assayed 2 or 3 weeks after injection of leukemic spleen cells, the colonies formed were comprised almost exclusively of myeloid cells. From spleen colonies formed from marrow or spleen cells obtained 1 week after the injection of leukemic spleen cells, all karyotypes contained 40 chromosomes, whereas from spleen colonies formed from marrow or spleen cells obtained 2 or 3 weeks after injection of spleen cells, almost all karyotypes contained 39 chromosomes. In contrast, most of the karyotypes found in spleen colonies formed from the injection of blood cells even 3 weeks after injection of leukemic spleen cells contained 40 chromosomes. All colonies containing cells with 39 chromosomes, leukemic colonies, contained only myeloid cells. We conclude that L-CFU-S differentiate only into the myeloid series. Early in the course of the disease there is an increase in both N-CFU-S and L-CFU-S in the spleen and marrow. As the disease progresses, the numbers of N-CFU-S in both spleen and marrow decline and, during the final week of the illness, the number of L-CFU-S in the marrow declines. The CFU-S in the peripheral blood are predominantly of normal type, even late in the disease when N-CFU-S are rare in the spleen and marrow.

Response of uremic patients to nandrolone decanoate.

The effect of anabolic steroid therapy on anemia in 13 women with and without kidneys undergoing chronic hemodialysis for renal failure was investigated. All but one of the six patients with kidneys demonstrated a noticeable increase in hematocrit level (6.4% to 14.6%). Of the anephric women, four of the seven also showed hematocrit level elevations, but these were less remarkable (3.1% to 6.4%). A majority of patients demonstrated increments in weight and serum creatinine measurements but no definitive alteration in serum blood urea nitrogen levels. The androgenic effects of nandrolone decanoate were minimal and well tolerated. We conclude that anabolic steroid therapy is effective in ameliorating the anemia of women undergoing hemodialysis, if given in a dose that produces minimal and tolerable untoward effects.

Residual marrow damage following therapy with cyclophosphamide.

Studies were performed to determine the type of residual marrow damage which occurs after injecting mice with 200 mg/kg of cyclophosphamide every 2 weeks for 5 courses. Mice treated with cyclophosphamide, and controls injected with normal saline, were studied 6 weeks after the last injection. Complete blood counts, and total nuclear cell counts from femoral marrow revealed no differences between the 2 groups. The number of CFUs in the marrow of cyclophosphamide treated mice was slightly, but significantly, lower than of controls. Cyclophosphamide treated and control mice were then exposed to 300 rad, and the rate of marrow CFUs recovery was determined. That of cyclophosphamide treated mice was significantly slower than that of controls. Stromal function of marrows from cyclophosphamide treated mice was significantly impaired. Also, however, the proliferative potential of marrow CFUs of cyclophosphamide treated mice was modestly reduced relative to that of controls. We conclude that cyclophosphamide treatment of mice results in significant residual marrow damage, due primarily to "stromal" damage, but also to decrease in the proliferative potential of CFUs.

Late effects of chemotherapy on hematopoietic progenitor cells.

Residual damage to marrow function has been observed in cyclophosphamide-treated or irradiated mice following recovery of marrow hematopoietic stem cells (CFU-S) and peripheral blood counts to pretreatment levels. Residual damage is evidenced by less rapid recovery of marrow CFU-S and blood counts following subsequent exposure to sublethal irradiation. Mice treated with busulfan also demonstrate residual marrow damage as evidenced by incomplete recovery of marrow CFU-S to pretreatment levels. We report here on studies to determine whether the residual marrow damage after radiation, cyclophosphamide, or busulfan therapy is exerted on the hematopoietic stroma's ability to support CFU-S proliferation (HS-P) or on the repopulating potential of the CFU-S per se. HS-P function is determined by measuring the ability of femora implanted subcutaneously into syngeneic mice to support the growth of host CFU-S. Evidence is presented to show that this function depends on fixed (nonmigrating) cells in the marrow environment. The repopulating potential of CFU-S is determined by measuring the rate of regeneration of marrow CFU-S after transplantation into lethally irradiated mice. The results of these studies indicate that exposure to 950 rad, busulfan, or cyclophosphamide all cause damage to the HS-P that persists for at least six weeks after therapy. After cyclophosphamide therapy, but not after exposure to the other two agents, HS-P function continues to improve six weeks after therapy and eventually reaches pretreatment levels. Only in busulfan-treated mice was the residual damage to the CFU-S repopulating capacity significantly more marked than damage to HS-P function.

Regulation of the plasma erythropoietin level in hypoxic rats.

Exposure to hypoxia results in an increase in the plasma erythropoietin (Ep) content to a peak level in 10-12 h. If hypoxia is discontinued before maximum plasma Ep concentrations are reached, then the plasma Ep level continues to rise before it declines toward normal. To clarify this phenomenon, we determined the Ep level in the plasma and kidneys at various times after rats that had been exposed to 0.42 atm returned to ambient pressure. The plasma Ep level 2 h after hypoxia was 4-5 times as high as it was immediately after hypoxia and then gradually declined. The Ep content of the kidneys rose for only 1 h after which it rapidly fell to an undetectable level. The most plausible interpretation of this data is that renal Ep production decelerates rapidly within 1 h after return to ambient pressure, but plasma Ep levels continue to rise because of the release of preformed Ep into the plasma. Experiments were also performed to determine whether an increased plasma Ep level inhibits the production or secretion of Ep. Three different types of experiments were performed in which the plasma Ep level was increased by intravenously injecting a large amount of Ep prior to, during, or after exposure to hypoxia and determining its effect on the plasma Ep activity from 15 min to 4 h after injection. In all experiments the plasma and renal Ep levels rose comparably in Ep-treated and control rats during exposure to hypoxia. The data do not support the hypothesis that a rise in the plasma Ep level inhibits Ep production or secretion into the plasma.

The influence of age and sex on erythropoietin titers in the plasma and tissue homogenates of hypoxic rats.

Experiments were performed to determine the erythropoietin (Ep) content of homogenates of kidneys and livers of male and female rats of various ages. In all studies, homogenates were adjusted to a concentration of 4 g of tissue per 12 ml of phosphate-buffered-saline, and the stimulus to Ep production consisted of exposure to 0.42 atmosphere for 4 h. The concentration of Ep in kidneys of male rats was about three times that found in those of females and was contained predominantly in the cortical portion of the kidneys. Ep was not detectable in kidneys of rats younger than 3 weeks of age, and reached a maximum concentration after 4 weeks of age. The Ep content of the liver was barely detectable regardless of the age of the rat or its plasma Ep titer; and did not increase significantly by administering angiotensin II or CCl4 (substances which increase extrarenal Ep production).

Effects of radiation on hematopoietic stroma.

The effects of x-irradiation on the CFU-S and on the hematopoietic stroma (HS-P) were studied and compared. Exposure to 300 rad reduced the CFU-S population to less than 1% of normal; by contrast more than 500 rad was required to detectably damage the HS-P. Although exposture to 500 rad did not detectably affect HS-P function, a second exposure to 500 rad as much as 4 weeks later did appreciably affect the HS-P. In addition, whereas the CFU-S returned to the pre-irradiation level within 6 weeks, the HS-P did not significantly recover. From these data we conclude that, with respect to the initial impact and exposure dose, HS-P is more radioresistant than CFU-S, but recovery from radiation induced damage of HS-P occurs slowly or does not take place.

Immunologically mediated aplastic anemia in mice: evidence of hematopoietic stromal injury and injury to hematopoietic stem cells.

Immunologically mediated aplastic anemia (AA) results when lymph node cells (LNC) from C3H/He mice are injected intravenously (i.v.) into H-2 identical CBA/J mice previously given 600 cGy sublethal total-body gamma irradiation (TBI). Previously, we showed that T lymphocytes injure pluripotent hematopoietic stem cells and cause severe pancytopenia and death in 80 to 100% of mice within 3 to 4 weeks, with changes in the bone marrow suggesting stromal injury. The following models were used to study the stroma: (1) Transplantation of femurs from AA mice into normal syngeneic CBA/J mice. After 6 weeks, colony-forming unit-spleen (CFU-S) levels in the femur implants were measured in both AA and control mice (600 cGy TBI only). (2) Development of Dexter long-term bone marrow cultures from AA and control mice, which were used to support hematopoietic bone marrow cells (colony-forming units-granulocyte/macrophage [CFU-GM]) from normal mice. (3) Cellulose ester membranes (CEM) were coated with hematopoietic stroma from AA and control mice and then implanted intraperitoneally (i.p.) into syngeneic CBA/J mice. Six months later, the CEM were removed and analyzed for the presence of trilineal hematopoiesis and bone. Injury to the hematopoietic stroma was documented by the following: (1) Femurs from AA mice had a decreased number of CFU-S compared to controls; (2) Dexter cultures from AA mice formed abnormal stromal layers with a decreased capacity to support CFU-GM from normal donor mice; and (3) CEM coated with stromal cells from AA mice had a decreased capacity to support trilineal hematopoiesis and bone compared to CEM coated with marrow stroma from control mice.

Hematopoiesis on cellulose ester membranes. XIII. A combination of cloned stromal cells is needed to establish a hematopoietic microenvironment supportive of trilineal hematopoiesis.

A mixture of stromal cells from murine bone marrow placed upon cellulose ester membranes (CEM) and then implanted intraperitoneally (i.p.) in mice results in a regenerated hematopoietic microenvironment which supports trilineal hematopoiesis. We used this model to study the capacity of 5 cloned murine stromal cell lines of marrow origin to support hematopoiesis in vivo: MBA-1 (fibroblast); MBA-2 (endothelial); MBA-13 (fibroendothelial); 14F1.1 (endothelial-adipose); and 14M1.4 (macrophage).10(7) stromal cells of a single cell line were applied to 1.5 cm2 CEM, which were folded into tubes and implanted i.p. into mice. Similarly, combinations of 4, 3 and 2 stromal cell lines were applied to CEM and implanted i.p. Single lines were implanted into syngeneic hosts of the same murine strain from which the clone was derived and into nude mice. Combinations of stromal cells were implanted only in nude mice to avoid allogeneic incompatibility. CEM implants were removed after intervals of 5 to 36 weeks and examined histologically. 1) Stromal cells of a single phenotype did not develop hematopoiesis. 2) A combination of 4 stromal phenotypes (MBA-1, MBA-2, MBA-13 and 14F1.1) formed a hematopoietic microenvironment supportive of trilineal hematopoiesis and bone. 3) The combination of 14F1.1 (endothelial adipose) + a second stromal phenotype--MBA-1 (fibroblast) or MBA-2 (endothelial) or MBA-13 (fibroendothelial) also supported trilineal hematopoiesis and bone. 4) CEM coated with MBA-13 or MBA-1 developed bone but no hematopoiesis. The endothelial-adipose phenotype appears to be essential to support hematopoiesis but requires other types of stromal cells--fibroblast, fibroendothelial or endothelial phenotype.

Busulfan-induced suppression of natural killer cell activity.

The effect of repeated injections of busulfan, an alkylating agent, on immune response of CAF1 mice was studied. A single injection of busulfan or acetone (vehicle to solubilize busulfan) acutely suppressed mitogenic and allogenic responses that normalized at two weeks. Repeated injections of busulfan (four injections), on the other hand, showed a transient suppression of the mitogenic responses. Natural killer (NK) activity during the first ten days after busulfan or acetone injections remained normal. NK activity diminished significantly after two injections of busulfan, remained low after four injections, and did not recover within four months of rest. Prolonged suppression of NK activity may be implicated as playing a role in the emergence of T-cell lymphomas in mice injected with busulfan.

Extraction of erythropoietin from kidneys.

Erythropoietin (Ep) in large amounts was detected in extracts of renal tissue from hypoxic rats. These extractions were performed by homogenization of kidney tissue in phosphate buffered saline, centrifugation at 3,000 g and collection of the supernate. Male kidneys contained more Ep than did females and the major portion of Ep is located in the renal cortex. Comparison of intrarenal and plasma Ep levels at various times following initiation and cessation of hypoxia appears to be a useful method for studying the kinetics of erythropoietin production and release, and also for studying feedback mechanisms that influence these functions.

Effects of bone marrow transplantation and polyinosinic-polycytidylic acid (poly I:C) on the rescue of animals from busulfan-induced NK suppression.

Repeated injections of busulfan (Bu) in CAF1 mice caused a long-lasting (greater than 16 weeks) decrease in their natural killer (NK) cell activity and impaired their resistance to transplantable lymphoma. Bu-treated mice had fewer spleen cells capable of binding to NK-sensitive YAC-1 target cells and reduced lymphokine-activated killer (LAK) cell activity as compared to normal age-matched controls. In contrast, interleukin 1 (IL-1) and interleukin 2 (IL-2) production were normal. Transplantation of normal bone marrow cells into Bu-treated mice resulted in an elevation of IL-2 production as well as in complete restoration of NK activity, target cell binding, and partial restoration of LAK activity. Resistance to transplantable lymphoma was equal to that of age-matched control mice. Polyinosinic-polycytidylic acid (Poly I:C) treatment resulted in immunomodulation in both control and Bu-pretreated mice. Twenty-four hours after Poly I:C injection, control and Bu-treated mice had higher levels of NK activity than did normal age-matched control mice, but the NK activity of Poly I:C/Bu-treated mice remained significantly lower than that of Poly I:C/control mice. The super-normal levels of NK activity in control and Bu-treated mice following Poly I:C administration were attributable, in part, to endogenous LAK activity. The generation of splenic LAK cells in vitro and target binding cells, which were reduced in Bu-pretreated mice, normalized following treatment with Poly I:C. Poly I:C treatment caused an increase in both IL-1 and IL-2 production in control and Bu-pretreated mice and in the ability of the treated mice to reject transplanted lymphoma cells. These results suggest that repeated injections of Bu decrease NK and LAK activity, but do not eliminate NK and LAK precursor cells. Thus, treatment with agents that increase IL-2 and/or interferon production can activate these cells to become effective killers and counter the long-lasting immunosuppressive effects of chemotherapy.

Normal colony stimulating factor (CSF) production by bone marrow stromal cells and abnormal granulopoiesis with decreased CFUc in S1/S1d mice.

The concentration of CFUC and the production of stromal-derived CSF in the femora of S1/S1d mice were determined. There was a lower concentration CFUC and a smaller total number of nucleated cells in the femoral marrow of WCB6. S1/S1d (S1/S1d) mice than in WCB6. +/+/(+/+) mice or in C57B1/6. +/+ (C57Bl) mice. On the other hand stromal-derived CSF production by femora from S1/S1d mice did not differ significantly from that of +/+'s. These observations indicate that the microenvironmental defect of S1/S1d mice results in decreased growth of granulocytic precursors as well as those of erythroid and megakaryocytic cells. This is consistent with the reported decrease in multipotential stem cell proliferation. Stromal cell derived CSF production was normal and could not be implicated in the decreased production of granulocytic precursors.

Acute interstitial nephritis in children: a report of 13 cases and review of the literature.

Clinical and pathologic data of 13 children, aged 5 to 16 years, with acute interstitial nephritis (AIN) are presented. The cause of AIN in these children was assessed as being related to infection in ten and methicillin in one; no infection, drug, or toxin could be implicated in two other patients. In addition to having various degrees of acute renal failure, all patients had systemic symptoms, most common of which were fatigue, fever, sore throat, and gastrointestinal disturbances. In six patients, the diagnosis of AIN was clinically suspected on the basis of tubular dysfunction such as low urinary specific gravity and glucosuria; in seven others the diagnosis was made after examination of the renal biopsy. Two patients had the nephrotic syndrome which resolved only after cytotoxic agents were added to corticosteroid therapy. The remaining 11 patients were given supportive therapy including peritoneal dialysis in one case. Complete recovery of renal function occurred in all patients within a mean interval of 69.5 +/- 34.7 days from the onset of symptoms, and all patients continue to have normal renal function during a follow-up period ranging from 1.5 to 10 years. We conclude that, in children, AIN is underdiagnosed, is most often associated with streptococcal infection, and carries an excellent prognosis.

The effect of cyclophosphamide on hematopoietic stem cells.

The effects of cyclophosphamide on hematopoietic stem cells (HSC) and on hematopoiesis was studied. Administration of 5 mg of cyclophosphamide to mice resulted in a rapid decrease in the number of HSC in the leg and spleen. The exocolonizing potential of HSC in the spleen recovered and reached supranormal levels in 7 days. That of HSC in the legs recovered to pretreatment values after 15 days. The endocolonizing potential of HSC in the leg, however, recovered in less than 4 days. The peripheral blood counts all dropped to a fraction of normal except for the platelet count, which was affected to a minimal degree. We suggest that cyclophosphamide irreparably damages most of the body's HSC. Those remaining in the leg then proliferate rapidly and repopulate other sites. HSC cannot, however, accumulate in the leg for more than a week after cyclophosphamide administration. Erythropoiesis does not recover until after this occurs. Platelets are least affected because of their resistance to the effects of the drug and because of the ability of megakaryocytes to increase their output of platelets without requiring differentiation of HSC into their compartment.