CX3CR1 deficiency leads to impairment of immune surveillance in the epididymis.

Mononuclear phagocytes (MPs) play an active role in the immunological homeostasis of the urogenital tract. In the epididymis, a finely tuned balance between tolerance to antigenic sperm and immune activation is required to maintain epididymal function while protecting sperm against pathogens and stressors. We previously characterized a subset of resident MPs that express the CX3CR1 receptor, emphasizing their role in antigen sampling and processing during sperm maturation and storage in the murine epididymis. Bacteria-associated epididymitis is the most common cause of intrascrotal inflammation and frequently leads to reproductive complications. Here, we examined whether the lack of functional CX3CR1 in homozygous mice (CX3CR1EGFP/EGFP, KO) alters the ability of MPs to initiate immune responses during epididymitis induced by LPS intravasal-epididymal injection. Confocal microscopy revealed that CX3CR1-deficient MPs located in the initial segments of the epididymis displayed fewer luminal-reaching membrane projections and impaired antigen capture activity. Moreover, flow cytometry showed a reduction of epididymal KO MPs with a monocytic phenotype under physiological conditions. In contrast, flow cytometry revealed an increase in the abundance of MPs with a monocytic signature in the distal epididymal segments after an LPS challenge. This was accompanied by the accumulation of CD103+ cells in the interstitium, and the prevention or attenuation of epithelial damage in the KO epididymis during epididymitis. Additionally, CX3CR1 deletion induced downregulation of Gja1 (connexin 43) expression in KO MPs. Together, our study provides evidence that MPs are gatekeepers of the immunological blood-epididymis barrier and reveal the role of the CX3CR1 receptor in epididymal mucosal homeostasis by inducing MP luminal protrusions and by regulating the monocyte population in the epididymis at steady state as well as upon infection. We also uncover the interaction between MPs and CD103+ dendritic cells, presumably through connexin 43, that enhance immune responses during epididymitis. Our study may lead to new diagnostics and therapies for male infertility and epididymitis by identifying immune mechanisms in the epididymis.

Metabolism of purines in macrophages. Effect of functional state of the cells.

Ecto-5'-nucleotidase is known to be diminished markedly in activated compared to control mouse macrophages. The level of three purine nucleoside metabolizing enzymes, adenosine deaminase (EC 3.5.4.4), purine nucleoside phosphorylase (EC 2.4.2.1), and adenine phosphoribosyltransferase (EC 2.4.2.7) were measured in the sonicates of different populations of mouse peritoneal macrophages. Levels of adenine phosphoribosyltransferase and purine nucleoside phosphorylase in macrophages that were elicited with sodium caseinate or activated in vivo by prior intravenous injection of Listeria monocytogenes were eight times higher than those in resident cells. Levels of adenosine deaminase also tended to increase and were two times higher in elicited cells than in resident cells. The Km of each enzyme was the same in each cell population. The findings suggest that the levels of the ecto-5'-nucleotidase and of the intracellular enzymes are coordinated.

Regulation of human eosinophil viability, density, and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fibroblasts.

Normodense human peripheral blood eosinophils were isolated under sterile conditions from the 22/23 and 23/24% interfaces and the cell pellet of metrizamide gradients. After culture for 7 d in RPMI media in the presence of 50 pM biosynthetic (recombinant) human granulocyte/macrophage colony-stimulating factor (rH GM-CSF), 43 +/- 7% (mean +/- SEM, n = 8) of the cells were viable; in the absence of rH GM-CSF, no eosinophils survived. The rH GM-CSF-mediated viability was concentration dependent; increased survival began at a concentration of 1 pM, a 50% maximal response was attained at approximately 3 pM, and a maximal effect was reached at concentrations of greater than or equal to 10 pM rH GM-CSF. In the presence of rH GM-CSF and mouse 3T3 fibroblasts, 67 +/- 6% (mean +/- SEM, n = 8) of the eosinophils survived for 7 d. In a comparative analysis, there was no difference in eosinophil viability after 7 and 14 d (n = 3) in the presence of 50 pM GM-CSF and fibroblasts. Culture with fibroblasts alone did not support eosinophil survival. The addition of fibroblast-conditioned media to rH GM-CSF did not further improve eosinophil viability, indicating a primary role for GM-CSF in supporting these eosinophil cell suspensions ex vivo and a supplementary role for 3T3 fibroblasts. Eosinophils cultured for 7 d localized on density gradient sedimentation at the medium/18, 18/20, and 20/21 interfaces of metrizamide gradients, indicating a change to the hypodense phenotype from their original normodense condition. In addition, the cultured eosinophils generated approximately 2.5-fold more LTC4 than freshly isolated cells when stimulated with the calcium ionophore A23187 and manifested sevenfold greater antibody-dependent killing of S. mansoni larvae than the freshly isolated, normodense cells from the same donor. Thus we demonstrate the rH GM-CSF dependent conversion in vitro of normodense human eosinophils to hypodense cells possessing the augmented biochemical and biological properties characteristic of the hypodense eosinophils associated with a variety of hypereosinophilic syndromes. In addition, these studies provide a culture model of at least 14 d suitable for the further characterization of hypodense eosinophils.

Interleukin 5 and phenotypically altered eosinophils in the blood of patients with the idiopathic hypereosinophilic syndrome.

We report that the hypodense eosinophil population in three patients with corticosteroid-unresponsive IHES was uniquely long lived ex vivo in the absence of exogenous cytokines. Serum or plasma from these patients conferred prolonged viability ex vivo to normodense eosinophils from reference donors and converted them to a functionally activated hypodense phenotype. In that antibody against IL-5 neutralized this activity in IHES serum, excessive quantities of this cytokine may account for the characteristic eosinophilia and long-lived, functionally augmented eosinophil phenotype in this disorder.

Concanavalin A induces microtubule assembly and specific granule discharge in human polymorphonuclear leukocytes.

Human neutrophils stimulated by concanavalin A (Con A, 100 microng/ml) contained markedly enhanced numbers of microtubules and discharged peroxidase-negative (specific) but not peroxidase-position (azurophile) granules. Release of lysozyme from specific granules was dose and time dependent, could be inhibitied by alpha-methyl-D-mannoside, and enhanced by cytochalasin B. Many microtubules were associated with internalized plasma membrane bearing Con A binding sites.

Particle concentration in the asteroid belt from pioneer 10.

The spatial concentration and size distribution for particles measured by the asteroid/meteoroid detector on Pioneer 10 between 2 and 3.5 astronomical units are presented. The size distribution is from about 35 micrometers to 10 centimeters. The exponent of the size dependence varies from approximately -1.7 for the smallest to approximately -3.0 for the largest size measured.

Eosinophils cocultured with endothelial cells have increased survival and functional properties.

Human peripheral blood eosinophils, cells often associated with allergic and parasitic diseases, were maintained in vitro for at least 14 days when they were cocultured with bovine endothelial cells and for at least 7 days when cultured with either bovine or human endothelial cell-derived conditioned medium. The cocultured eosinophils became hypodense and generated about three times as much leukotriene C4 upon activation with calcium ionophore and killed about three times as many antibody-coated larvae of Schistosoma mansoni as freshly isolated normodense eosinophils. That these cells can be maintained in vitro by coculture with endothelial cells, and the surprising finding that the cocultured eosinophils have biochemical, cytotoxic, and density properties similar to those of eosinophils in patients with allergic and other disorders, will facilitate investigation of the regulation and role of these cells in health and disease.

Activation energy for water diffusion across the toad bladder: evidence against the pore enlargement hypothesis.

The activation energy (E(A)) for the diffusion of water across the epithelial cell layer of the toad bladder was determined in the absence and presence of vasopressin. An experimental approach was employed which minimized the effects of unstirred layers and the thick supporting layer of the bladder on the measurement of water diffusion. E(A) in the absence of vasopressin was 11.7 +/-1.4 kcal.mole(-1); after vasopressin it was 10.6+/-1.1 kcal.mole(-1). The difference between the two values was not significant. The results are consistent with an increase in the number rather than the size of aqueous channels in the cell membrane, a finding which differs from the generally held view that the hormone increases the radius of pores in the membrane.

Properties of highly purified leukotriene C4 synthase of guinea pig lung.

Leukotriene C4 (LTC4) synthase, which conjugates LTA4 and LTA4-methyl ester (LTA4-me) with glutathione (GSH) to form LTC4 and LTC4-me, respectively, has been solubilized from the microsomes of guinea pig lung and purified 91-fold in four steps to a specific activity of 692 nmol/10 min per mg protein using LTA4-me as substrate. LTC4 synthase of guinea pig lung was separated from microsomal GSH S-transferase by Sepharose CL-4B chromatography and further purified by DEAE-Sephacel chromatography, agarose-butylamine chromatography, and DEAE-3SW fast-protein liquid chromatography. It was also differentiated from the microsomal GSH S-transferase, which utilized 1-chloro-2,4-dinitrobenzene as a substrate, by its heat lability and relative resistance to inhibition by S-hexyl-GSH. The Km value of guinea pig lung LTC4 synthase for LTA4 was 3 microM and the Vmax was 108 nmol/3 min per microgram; the Km values for LTA3 and LTA5 were similar, and the Vmax values were about one-half those obtained with LTA4. The conversion of LTA4-me to LTC4-me was competitively inhibited by LTA3, LTA4, and LTA5, with respective Ki values of 1.5, 3.3, and 2.8 microM, suggesting that these substrates were recognized by a common active site. IC50 values for the inhibition of the conjugation of 20 microM LTA4-me with 5 mM GSH were 2.1 microM and 0.3 microM for LTC4 and LTC3, respectively. In contrast, LTD4 was substantially less inhibitory (IC50 greater than 40 microM), and LTE4 and LTB4 had no effect on the enzyme, indicating that the mixed type product inhibition observed was specific for sulfidopeptide leukotrienes bearing the GSH moiety.

Human eosinophils have prolonged survival, enhanced functional properties, and become hypodense when exposed to human interleukin 3.

Human eosinophils were cultured in the presence of recombinant human IL-3 for up to 14 d and their biochemical, functional, and density properties were assessed. After 3 d of culture in 10 pM IL-3, eosinophils had a viability of 70% compared with only 10% in enriched medium alone. Neither IL-1 alpha, IL-2, IL-4, tumor necrosis factor, basic fibroblast growth factor, nor platelet-derived growth factor maintained eosinophil viability. The 7- and 14-d survival of the cultured eosinophils was 55 and 53%, respectively. No other cell type, including neutrophils, was present after culture. After 7 d of culture, the normodense eosinophils were converted to hypodense cells as assessed by density centrifugation. Eosinophils exposed to 1,000 pM IL-3 for 30 min or cultured in 10 pM IL-3 for 7 d generated approximately threefold more leukotriene C4 (LTC4) in response to calcium ionophore than freshly isolated cells. Furthermore, whereas freshly isolated eosinophils killed only 14% of the antibody-coated Schistosoma mansoni larvae, these eosinophils killed 54% of the larvae when exposed to 100 pM IL-3. The enhanced helminth cytotoxicity was maintained for 7 d when eosinophils were cultured in the presence of both 10 pM IL-3 and 3T3 fibroblasts, but not when eosinophils were cultured in the presence of IL-3 alone. IL-3 thus maintains the viability of eosinophils in vitro, augments the calcium ionophore-induced generation of LTC4, enhances cytotoxicity against antibody-sensitized helminths, and induces the eosinophils to become hypodense cells. These phenotypic changes in the eosinophil may be advantageous to host defense against helminthic infections but may be disadvantageous in conditions such as allergic disease.

Long-term effects of pancreatic transplant function in patients with advanced juvenile-onset diabetes.

Present methods of management of juvenile-onset diabetes mellitus do not prevent serious and debilitating complications affecting multiple organ systems. In an effort to reverse advanced forms of these complications, segmental transplantation of the pancreas has been performed on 10 patients, seven of whom simultaneously or subsequently received renal transplants. Long periods of normoglycemia (two to four and one-half years) were achieved in two patients who also maintained transplant kidney function. The course of these two patients is described to illustrate the possible value and limitations of the procedure. These patients had normal blood glucose levels, exhibited repeated normal intravenous glucose tolerance curves, and had repeated normal endogenous insulin levels. Their courses were characterized by (1) absence of problems related to pancreatic exocrine secretions into the bladder; (2) stable eye changes despite some episodes of hemorrhage from preexisting retinopathy; (3) vascular complications, including stroke and gangrene of extremities necessitating amputation despite successful femoropopliteal bypass grafting; (4) peripheral neuropathy; and (5) repeated infections. Both patients succumbed to vascular complications. Thus, pancreatic transplantation can maintain blood glucose and insulin at normal levels for extended periods of time. However, it does not reverse such complications as advanced retinopathy or atherosclerosis. Since the procedure may have value in preventing progression of these complications, it should be evaluated in patients with less advanced complications of diabetes.

Individualization of immediate posttransplant immunosuppression. The value of antilymphocyte globulin in patients with delayed graft function.

In patients with delayed graft function (DGF), the use of cyclosporine (CsA) has been reported to prolong DGF, increase the number of required dialyses, increase the duration of hospitalization, and be associated with decreased graft survival. Routine postoperative antilymphocyte globulin (ALG) use has been advocated, but ALG is associated with increased viral infection. We studied outcome of individualization of immunosuppression. Between 11/84 and 8/86, first-cadaver transplant recipients whose serum creatinine (Cr) fell greater than or equal to 30% in the first 24 hr (immediate function) were started on CsA and prednisone (P) (group 1, n = 26). The remainder were randomized to P and azathioprine (group 2, n = 32) or P and ALG (group 3, n = 26), and switched to CsA when serum Cr fell greater than 30% (minimum 5 days ALG for the ALG group). P taper was the same in all groups. Patients with DGF (groups 2 and 3) had longer preservation time and higher peak PRA (P less than .05) than group 1. Groups were otherwise equivalent. One and 2-year patient survival was 96% (3 cardiovascular deaths; all with functioning grafts). One-year graft survival was 87% for group 1, 87% for group 2, and 82% for group 3(NS). In patients requiring dialysis, mean day off dialysis was 12 +/- 3 in both groups 2 and 3. Mean hospital stay was 12.5 +/- 1.3 days for group 1, 21.6 +/- 2.1 days for group 2 (P less than .05 vs. 1 & 3), and 14.5 +/- 1.2 days for group 3 (NS vs. 1). The increased hospital stay for group 2 patients was mainly due to increased in-hospital rejections: 75% for group 2, (P less than .05 vs. group 1 [35%], and group 3 [11.5%]). In addition, more group 2 in-hospital 1st rejections were steroid resistant as compared to group 1; 46% group 1 patients have remained rejection free as compared to 0% group 2 (P less than .05 vs. 1 and 3) and 35% of group 3 (P less than .05 vs. 1 and 2). Mean serum creatinine at 6-12 months remained higher in patients with DGF (group 1 P less than .05 vs. 2 and 3). Rejection was the major cause of graft loss in all groups.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of the referring dialysis center on renal transplant results.

Between 1/1/76 and 12/31/86, 448 patients underwent transplantation (360 first transplants). Of these, 286 (230 first) were referred by 5 dialysis centers, each referring more than 40 recipients. The remainder were referred by a large number of centers. Using our 5 largest referral centers, we studied the effect of dialysis center on graft and patient survival. There was no difference between dialysis centers in patient survival. Actuarial graft survival differed significantly for all cadaver transplants and for first cadaver transplants (P less than 105). Significant differences persisted when groups were subdivided by type of immunosuppression (azathioprine vs cyclosporine). Demographic (age, race, cause of renal disease) and immunologic (transfusions, PRA, matching) differences between groups did not explain the difference in graft survival. We conclude that referring dialysis center is a previously unrecognized factor affecting transplant outcome. Further studies with larger numbers will be required to determine the underlying reasons for ths phenomenon.

Treatment of renal transplant rejection episodes in patients receiving prednisone and azathioprine. A cost-effective approach.

Antilymphocyte globulin (ALG) has been advocated for the treatment of renal transplant rejection episodes in patients maintained on prednisone and azathioprine. Treatment with steroids (outpatient) is considerably less expensive than with ALG (inpatient), so we studied whether routine ALG was necessary. Between 3/82 and 11/83, 54 cadaver transplant recipients maintained on prednisone and azathioprine who developed a first rejection episode were randomized to receive--for treatment of their first, and if necessary second, rejection--methylprednisolone (MP) plus ALG (n = 24), or MP alone, with ALG added if treatment failed (n = 30). Treatment failure was defined as continuing deterioration on T131 iodohippuran scan, rising serum creatinine level, or lack of improvement within 7 days. There was no significant difference in patient survival, graft survival, mean number of rejections, and infection rate between the two groups: 60% (18/30) of first and 50% (10/10) of second rejection episodes responded to MP alone. We conclude that patients are not penalized by initial rejection treatment with MP. Many rejection episodes respond to steroids alone; elimination of routine ALG use will save hospitalization time and expense.

ALG treatment of steroid-resistant rejection in patients receiving cyclosporine.

Thirty-one episodes of biopsy-proved acute rejection (R) in 28 patients maintained on cyclosporine did not respond to high-dose steroids and were treated with antilymphocyte globulin (ALG). Cyclosporine was discontinued in all but three during ALG administration. (A) Twenty-four patients received 26 courses of ALG within 90 days of transplant (11 1st R, 15 2nd or 3rd). Seven treatment courses were cut short due to infection (4), ongoing R (2) and a combination of infection and rejection (1). Only 1 of 7 has a functioning graft. Of the remaining 19 full ALG courses (17 patients) (8 1st R, 11 2nd or 3rd), 13 (11 patients) responded (7 1st R, 6 greater than 1st). The remaining 6 patients lost their grafts to ongoing acute rejection. (B) Five patients were treated after 6 months posttransplant; two responded but no grafts currently function. (C) Overall 7 patients developed systemic infection (7 viruses, 1 Candida) with 1 death, and 2 additional patients developed severe thrombocytopenia and leukopenia. Patients responding to their ALG course were restarted on cyclosporine. We conclude that ALG is not as effective in reversing steroid-resistant rejection in patients maintained on cyclosporine as it has been in patients maintained on azathioprine. However, more than 50% of steroid-resistant rejection episodes are reversed.

Successful transplantation after conversion of a positive crossmatch to negative by dissociation of IgM antibody.

Preliminary crossmatching usually eliminates highly sensitized patients from consideration for renal transplantation. However, if the crossmatch is positive because of the presence of IgM antibody, this activity can be eliminated by treatment with the reducing agent Dithiothreitol (DTT). Successful transplantation may then be possible in patients whose crossmatch is positive due to the presence of IgM antibody. After treatment with DTT, the sera of 25 highly sensitized patients were measured for cytotoxicity against a selected panel of 40 cells. Those whose high %PRA could be attributed to blood transfusions or previous transplants did not change with DTT. Only two patients who had developed high panel reactivity, without a clear cause, had little reactivity remaining after DTT treatment of their sera. To select patients whose crossmatch might be rendered negative by DTT treatment, we developed a "minipanel" screening protocol. Patients whose monthly PRA cells increased greater than 30% from baseline had their serum samples treated with DTT to reduce IgM. The treated sera were tested against a panel of six cells. If there was little or no cytotoxicity, it was assumed that IgM antibody was responsible for the positive crossmatches. All subsequent cadaver donor crossmatches were done with and without DTT treated sera. Five patients (2 living-related; 3 cadaver) with current crossmatches positive before, but negative after, DTT treatment continue to have functioning kidneys 3-15 months after renal transplantation. There were no hyperacute rejections. We conclude that patients with IgM antibody can be successfully transplanted if they have a negative cross-match after reduction of IgM antibody in their serum samples. A "minipanel" helps to identify patients who will benefit from DTT treatment.

Percutaneous transluminal dilation in renal transplant arterial stenosis.

Twelve hypertensive patients underwent percutaneous transluminal dilation (PTD) for relief of arterial stenosis complicating renal allotransplantation. Two patients underwent repeat PTD for recurrent stenosis and hypertension. Six patients had end to end anastomosis of the donor renal artery to the recipient hypogastric artery; four of six PTDs were successful. Six patients had end to side anastomosis of the donor renal artery to the recipient external iliac artery; seven of eight PTDs, including one of two repeat PTDs, were successful. Prior to PTD, all patients were using several antihypertensive medications. Following successful PTD, the mean blood pressure dropped from 184 +/- 15/118 +/- 9 to 133 +/- 13/89 +/- 11 mm Hg (P < 0.001) and remained at that level for up to 15 months (average followup 9 months) with decreased or no antihypertensive medications. Since surgical correction of arterial stenosis occurring after renal transplantation is difficult and may endanger the graft, PTD should be the first interventional therapy.

The value of needle renal allograft biopsy. III. A prospective study.

Previous studies of the value of percutaneous renal transplant biopsy have been retrospective. We prospectively studied whether biopsy affected patient management. Thirty-five patients with elevated serum creatinine level underwent 44 biopsies in situations in which the diagnosis was in doubt. At the time of biopsy, all clinical and laboratory data were reviewed, and a proposed treatment plan was outlined. Biopsy results were available within 24 hours. We evaluated whether biopsy influenced treatment. Other than hematuria (less than 24 hours), there were no complications. Nine biopsy specimens (20.5%) were inadequate for evaluation. Forty-six percent of adequate biopsy specimens (36% of total biopsy specimens) influenced patient management. Adequate biopsy specimens resulted in a change in treatment in 10 of 19 patients receiving prednisone and azathioprine and 6 of 16 receiving prednisone and cyclosporine. The remaining biopsy specimens, although not changing management confirmed the treatment plan in ambiguous clinical situations. We conclude that percutaneous biopsy is an important aid in patient management.

Definition, diagnosis, and management of rejection in the second to sixth months posttransplant--an overview.

CsA immunosuppression has resulted in decreased graft loss from rejection. However, rejection episodes do occur and, in fact, rejection remains as the major cause of graft loss in the CsA-treated patient. CsA, itself, has added to the differential diagnosis of renal dysfunction following transplantation. In the majority of circumstances, rejection can be differentiated from CsA nephrotoxicity as well as other causes of renal dysfunction by a combination of clinical presentation, renal scan and sonography, CsA levels, and percutaneous allograft biopsy. In some circumstances, a therapeutic trial of lowering the CsA dose may be indicated before extensive laboratory study. Most acute rejection episodes will respond to increased steroid doses. In patients with low CsA levels, increasing the CsA dose may be advised. Steroid-resistant rejection frequently responds to ALG. Patients with repeated episodes of renal dysfunction may be stabilized by using the combination of prednisone, azathioprine, and CsA.