Gene targeting in normal somatic cells: inactivation of the interferon-gamma receptor in myoblasts.

Gene targeting in somatic cells represents a potentially powerful method for gene therapy, yet with the exception of pluripotent mouse embryonic stem (ES) cells, homologous recombination has not been reported for a well characterized, non-transformed mammalian cell. Applying a highly efficient strategy for targeting an integral membrane protein--the interferon gamma receptor--in ES cells, we have used homologous recombination to target a non-transformed somatic cell, the mouse myoblast, and to compare targeting efficiencies in these two cell types. Gene-targeted myoblasts display the properties of normal cells including normal morphology, ability to differentiate in vitro, stable diploid karyotype, inability to form colonies in soft agar and lack of tumorigenicity in nude mice.

Reduced systemic drug exposure by combining intraarterial cis-diamminedichloroplatinum(II) with hemodialysis of regional venous drainage.

During cancer chemotherapy toxicity to normal tissues often limits the tolerable dose. To increase drug delivery to tumor while maintaining tolerable systemic exposure, regional treatments, such as intraarterial drug delivery, have been used. Despite intraarterial delivery, systemic toxicity often remains the dose-limiting sensitivity. If systemic drug exposure could be reduced after intraarterial infusion, the intraarterial dose could be increased, which should increase the therapeutic response. We compared the pharmacokinetic advantage after cisplatin infusion into the internal carotid artery to that obtained after infusing cisplatin into the internal carotid artery during extracorporeal removal of cisplatin from the jugular blood by hemodialysis. Four patients with malignant gliomas received intracarotid cisplatin, 100 mg/m2 over 60 min, every 4 weeks. During one treatment, while cisplatin was infused into the internal carotid artery, the jugular blood was dialyzed extracorporeally at 300 ml/min and returned to the inferior vena cava. Seventy to 96% of the free platinum that entered the dialyzer was removed. By aspirating blood from the jugular vein at 300 ml/min, 30-79% of the ipsilateral carotid blood was collected for extracorporeal circulation. Hemodialysis of the cerebral venous drainage during intracarotid infusion reduced the systemic exposure to cisplatin by 51-61% when compared to the exposure from internal carotid artery infusion without hemodialysis. The pharmacokinetic advantage (brain/body exposure ratio) was increased from 3 to 5/1 during internal carotid artery infusion alone to as much as 15/1 during treatment combining intracarotid infusion with hemodialysis of the jugular blood. Systemic toxicity now limits the dose of cisplatin that can be administered safely. Increased tumor exposure without increased systemic toxicity may be possible with the technique described and greater doses of cisplatin. Assuming no associated local toxicities, the results of the current study indicate that the dose of intracarotid cisplatin can be increased while maintaining tolerable systemic exposure.

Severe glomerulonephritis with late emergence of classic Wegener's granulomatosis. Report of 4 cases and review of the literature.

We have analyzed an unusual group of 19 patients (15 previously reported) with Wegener's granulomatosis, who presented with severe glomerulonephritis and developed diagnostic respiratory lesions only after 4 to 78 months. Necrotizing glomerulonephritis, often with crescents, and rarely with vasculitis, was the predominant renal lesion. Wegener's granulomatosis was unsuspected initially, since systemic manifestations, such as fever, arthralgias, malaise, and even pulmonary hemorrhage, were nonspecific or transient, and because renal biopsy findings resembled those seen in microscopic polyarteritis or idiopathic crescentic nephritis. Despite therapy, usually with corticosteroids, only 4 patients maintained adequate renal function. Most patients were receiving chronic dialysis when respiratory involvement developed. Cavitary nodular pulmonary infiltrates were seen in 12 of the 17 patients with lung involvement, and otorhinological disease occurred in 10 patients. Arthralgias, fever, and cough, with or without hemoptysis, were common. Wegener's granulomatosis was diagnosed by lung biopsy in 15 cases and by nasal biopsy in 4. Specific treatment was required for the respiratory disease and was delayed in many patients, because of lack of awareness that Wegener's granulomatosis may present with primary glomerulonephritis and become active during chronic renal failure or dialysis. Nevertheless, all but 1 patient eventually responded to treatment, although 3 additional patients died of late complications.

Therapy of anti-glomerular basement membrane antibody disease: analysis of prognostic significance of clinical, pathologic and treatment factors.

We have compared the effect of therapy with immunosuppression alone to immunosuppression plus plasma exchange on the clinical course and rate of disappearance of antibody in 17 patients with anti-glomerular basement membrane (anti-GBM) antibody-induced renal disease. Patients receiving immunosuppression (n = 9) and those receiving plasma exchange (n = 8) were similar in terms of entry clinical characteristics, pulmonary manifestations and complications associated with therapy. Rate of disappearance of anti-GBM antibody as estimated from serial estimates of antibody binding was significantly more rapid in patients receiving plasma exchange, and mean serum creatinine in these patients at end of therapy was half that of the patients receiving immunosuppression alone. Analysis of clinical and pathologic values at study entry, however, indicated that the percent of crescents on initial renal biopsy and entry serum creatinine correlated better with outcome than did therapeutic modality. Thus, though plasma exchange may offer some advantage over immunosuppression alone in the treatment of this disease, degree of pathologic involvement appears to be the major factor affecting outcome. Patients with low cresents (less than 30%) and well preserved function did well with either treatment, while patients with severe crescentic involvement and impaired glomerular filtration rate did poorly.

Renal tubular function in cyclosporine-treated patients.

Renal tubular function was studied in 32 consecutive patients treated with cyclosporine for autoimmune uveitis. Cyclosporine dosage was modified to maintain the serum creatinine level at less than 2 mg/dl. No evidence of aminoaciduria, glucosuria, hypokalemia, hypophosphatemia, or hypouricemia indicative of the Fanconi syndrome was detected during three to six months of observation. The renal tubular reabsorption of phosphate, determined by renal threshold phosphate concentration (TmPO4/GFR), remained unchanged. An early decrease in the serum magnesium level was accompanied by a decrease in daily urinary magnesium excretion. Concurrent with the development of a mild reduction of the glomerular filtration rate, the fractional excretion of magnesium was slightly higher and that of calcium was lower than pretreatment values. There were modest elevations of serum uric acid and potassium values. Furthermore, lysozymuria was not observed. Thus, the alterations in tubular functions observed in our cyclosporine-treated patients are comparable to those associated with mild chronic renal insufficiency of various causes and are not specific manifestations of cyclosporine tubular toxicity.

Prognostic factors in lupus nephritis. Contribution of renal histologic data.

The predictive value of laboratory results and renal histologic data was examined in 102 patients upon entry into prospective, randomized, therapeutic trials of lupus nephritis. Three clinical features at the time of entry into the study were individually associated with increased rates of renal failure: age less than 24 years, male gender, and an elevated serum creatinine level. Subjects with diffuse proliferative or membranoproliferative glomerulonephritis were at a modest but significantly increased risk for the development of end-stage renal disease compared with patients with other classes of lupus nephritis. Semiquantitative scores of histologic features (specified by activity and chronicity indexes) identified subgroups of patients with comparatively high renal failure rates. To address the controversial issue of whether renal histologic data significantly improve the outcome predictions in patients with lupus nephritis, multivariate survival models were generated, permitting simultaneous consideration of multiple prognostic factors. Outcome predictions based on the strongest clinical predictors (age, sex, and serum creatinine level) were significantly enhanced by the addition of activity and chronicity indexes. Only age and chronicity index contributed significantly to the five-variable model and together constituted a two-variable model, the predictions of which were similar to observed outcomes. In the context of the highly significant prognostic indicators (age and chronicity index), immunosuppressive agents appeared to provide a slight therapeutic advantage over oral corticosteroids alone.

Treatment of lupus nephritis.

Patients with lupus nephritis pose a therapeutic challenge and stimulate investigation of innovative treatment strategies. Although patient survival and renal function outcomes have improved over the last 4 decades, contemporary immunosuppressive regimens are not consistently effective and often require extended courses associated with insidious toxicities. Several strategies are under investigation to induce remissions more rapidly and to reduce the risk of long courses of cytotoxic drug therapy. The combination of pulse methylprednisolone and pulse cyclophosphamide may be more effective than pulse cyclophosphamide alone for patients with relatively severe proliferative lupus nephritis. Ongoing clinical studies evaluate the risk/benefit of other intensive induction regimens (eg, combination fludarabine with relatively low-dose pulse cyclophosphamide). A particularly vigorous strategy employs immunoablative cyclophosphamide with or without stem cell rescue. Several studies of sequential immunosuppressive therapy are in progress. It is anticipated that long-term toxicities can be lessened by substituting various maintenance agents (eg, azathioprine or mycophenolate mofetil) after initial cyclophosphamide therapy has induced a renal response. Additional information is needed to determine the role of this strategy. Furthermore, a number of standard and experimental immunosuppressive regimens (that do not include cyclophosphamide) are under investigation as well. Innovative approaches (eg, costimulatory blockade) offer the hope of more effective treatments without the risks of contemporary regimens.

Natural history and treatment of lupus nephritis.

Renal involvement occurs in the majority of patients with systemic lupus erythematosus. Contemporary therapeutic regimens for immunosuppression and for the treatment of hypertension, hyperlipidemia, infections, and seizures have likely contributed to improvements in the prognosis of these patients over the last four decades. Corticosteroids usually ameliorate the manifestations of lupus nephritis but achieve less complete and sustained remissions than do cytotoxic drugs. Among the cytotoxic drugs, pulse cyclophosphamide has one of the best profiles of efficacy and toxicity. Because each episode of exacerbation of lupus nephritis results in cumulative scarring, atrophy and fibrosis, we recommend continued maintenance treatment for 1 year beyond the point of complete remission of proliferative lupus nephritis. Studies are in progress to determine whether innovative treatment strategies will enhance efficacy and minimize toxicity associated with cytotoxic drug therapies. Lupus membranous nephropathy poses a lower risk of renal failure, but persistent nephrotic syndrome confers risks of cardiovascular events; this form of lupus nephritis is usually treated with less intensive regimens of corticosteroids, cytotoxic drugs, or cyclosporine. The prognosis and overall success of treatment for lupus nephritis seem to vary widely among geographically and racially diverse populations. The causes for the apparently worse prognosis and poorer responses to treatment of lupus nephritis in Black patients are currently unexplained and require further study. Until such data are available, caution is clearly warranted in extrapolating evidence, particularly about prognosis and effects of treatment, among different populations of patients with lupus nephritis.

Treatment of lupus nephritis.

Lupus nephritis is a prototype of immune complex-mediated glomerulonephritis. A broad range of clinical presentations and histological changes (proliferative, membranous, or both) are observed. Patients are at risk for progressive renal function deterioration as a result of the interaction of various active immunologic and chronic sclerosing mechanisms of kidney injury. Hypertension and hyperlipidemia contribute to morbidity and mortality. Monitoring serological parameters, urinary protein excretion rate and, especially, the urinary sediment facilitate the prompt recognition and treatment of this disorder. Kidney biopsy evaluation often clarifies the type, severity, and potential reversibility of the underlying renal lesions. Although contemporary immunosuppressive regimens for proliferative lupus nephritis have reduced the risk of end-stage renal failure, they are potentially toxic and not universally effective. Decisions regarding the intensity and duration of these treatments are difficult and are based on the severity of the disease, the initial response to therapy, and the risk for drug-induced toxicities. Studies are in progress to evaluate alternative regimens for proliferative lupus nephritis and membranous lupus nephropathy.

New prospects for treatment of lupus nephritis.

Systemic lupus erythematosus (SLE) is envisioned to arise from hyperactivate helper T-cells that cause polyclonal B-cell secretion of pathogenic autoantibodies and formation of immune complexes which deposit in sites such as the kidney. The most widely used immunosuppressive drugs, notably corticosteroids and cyclophosphamide, are often criticized as being nonspecific. In fact, these agents may be effective in SLE and lupus nephritis because broad, rather than highly selective, effects are required to control the aberrant immune system. Nonetheless, these agents are not uniformly effective and are associated with substantial toxicities. The lack of universal efficacy raises the specter that lupus is a heterogeneous disorder with different etiopathogenesis in different subsets of patients (as in lupus-prone mice). Therapeutic prospects for the upcoming millennium include new forms and combinations of chemotherapeutic agents (mycophenolate and adenosine analogues), attempts to achieve immunological reconstitution using near-ablative chemotherapy (with or without bone marrow or stem cell rescue), monoclonal antibodies, and other inhibitors of T-cell costimulatory pathways (e.g., anti-CD154 and/or CTLA4-Ig). The prospect for gene therapy has already been realized in some animal models of SLE. In human SLE, the feasibility of gene therapy will depend on further definition of lupus-promoting genes and availability of methods to establish stable expression of potentially corrective genes.

Renal disease in systemic lupus erythematosus.

Lupus nephritis is the archetype of diseases caused by the deposition of immune complexes. The characteristics which render only a portion of circulating immune complexes nephritogenic are not well delineated, but cat-ionic charge of antigen and/or antibody may contribute. Lupus nephritis is characterized by extreme diversity of clinical manifestations and pathologic features. Scrupulous monitoring of urinary sediment and renal function tests is necessary to identify renal involvement in a phase which is amenable to therapeutic intervention. Evaluation of renal biopsies also assists in development of indications for therapy. The pathology of lupus nephritis is classified primarily as mesangial, focal proliferative, diffuse proliferative, and membranous nephropathy. Indexes of activity and of chronicity provide a succinet description of the balance of reversible and irreversible disease. The activity index incorporates glomerular hypercellularity, karyorrhexis/fibrinoid necrosis, leucocyte exudation, hyaline thrombi, cellular crescents, and interstitial inflammation. The chronicity index incorporates glomerular sclerosis, fibrous crescents, tubular atrophy, and interstitial fibrosis. Delineation of the activity and chronicity indexes also facilitates the assessment of prognosis and the ordering of indications for therapy.

Studies of immunosuppressive drugs in the treatment of lupus nephritis.

Long-term, controlled trials to evaluate the efficacy and toxicities of immunosuppressive drug therapies compared to treatment with corticosteroids alone in the management of patients with lupus nephritis have been performed. The studies demonstrate that immunosuppressive drug regimens, particularly intravenous cyclophosphamide, reduce the likelihood of end-stage renal failure. However, toxicities such as herpes zoster and ovarian failure were found to be increased in patients with cyclophosphamide.

Combined use of cyclosporine and ketoconazole in the treatment of endogenous uveitis.

Ten patients with endogenous uveitis were in clinical remission attributable to treatment with cyclosporine and prednisone. After the cyclosporine dose was reduced by two thirds, these patients were randomly assigned to treatment with or without ketoconazole, a potent inhibitor of cytochrome P-450, in a double-masked placebo-controlled study. The dose was reduced over three days. During a three-month follow-up, no patients treated with ketoconazole had a relapse of uveitis, while four of six (66%) control subjects had a flare-up. Toxicity in the ketoconazole-treated group was limited to a transient decrease in glomerular filtration rate (20% from baseline) at one month in two of six (33%) patients. Renal function was stabilized by further reduction of the cyclosporine dose.

Metabolic and renal effects of interleukin-2 immunotherapy for metastatic cancer.

The systemic administration of recombinant interleukin-2 (IL-2) either alone or in combination with lymphokine activated killer cells is a new approach to the immunotherapy of metastatic cancer in man. Renal toxicity is often a dose-limiting side effect of IL-2 administration. This prospective study of 17 consecutive patients receiving parenteral high dose IL-2 documents a reversible syndrome of hypotension, oliguria, fluid retention, azotemia and very low urinary excretion of sodium (median FeNa of 0.04%). The median nadir urinary uric acid to urinary creatinine ratio during IL-2 therapy was 0.2. This IL-2 regimen induces a reversible renal hypoperfusion syndrome (pre-renal azotemia) without evidence of acute uric acid nephropathy. Hypophosphatemia [median serum phosphorus of 1.9 mg/dl (0.61 mmol/l)] prompted further study of tubular function. Urinary excretions of phosphorus, calcium and magnesium were very low. Arterial blood gases revealed hyperventilation without alkalemia. The hypophosphatemia probably reflects increased utilization of inorganic phosphorus by rapidly proliferating lymphoid cells.

A pilot study of low-dose fludarabine in membranous nephropathy refractory to therapy.

BACKGROUND: Lymphocytes are believed to play a role in the induction and perpetuation of membranous nephropathy. Fludarabine is a purine nucleoside analog with selective activity against both dividing and resting lymphocytes. We evaluated the tolerance, toxicity, pharmacokinetics, immunologic, and clinical effects of fludarabine in patients with membranous nephropathy in an single arm pilot study. PATIENTS AND METHODS: Eight patients with idiopathic (n = 7) or lupus (n = 1) membranous nephropathy who had failed high-dose prednisone (n = 8) and/or alkylating agents (n = 2), or cyclosporine (n = 1) were treated with 6-monthly cycles of fludarabine (cycles 1-2, 20 mg/m2/day x 2 days, cycles 3-6, 20 mg/m2/day x 3 days). Mean proteinuria was 9 g/day with a mean duration of disease of 25 months (range 12-48). Proteinuria, GFR and effective renal plasma flow were compared before and after completing the treatment. RESULTS: Seven patients completed the protocol. CD3, CD4, CD8 and B cell counts decreased by 53%, 46%, 61% and 84%, respectively, at the end of treatment and remained at lower than pretreatment levels 6 months after completing the trial. Despite lymphopenia, serum immunoglobulin levels remained unchanged. Both naive (CD45RA+) and memory CD4+ T cells (CD45RO+) were reduced (naive > memory). Proteinuria decreased by > or = 50% in 5 out of 7 patients (p = 0.11). Filtration fraction improved in all patients with decreased filtration fraction at baseline. The only side-effect observed was one episode of acute bacterial sinusitis that responded promptly to antibiotic therapy. CONCLUSION: We conclude that low-dose fludarabine treatment in patients with membranous nephropathy is well tolerated and results in significant lymphopenia involving B more than T cells. In this pilot study improvement in proteinuria and filtration rate were observed. Additional studies are required to determine the optimal dose and clinical efficacy of fludarabine.

Angiotensin I converting enzyme gene polymorphisms in systemic lupus erythematosus: decreased prevalence of DD genotype in African American patients.

The presence of the D (deletion) allele at the angiotensin converting enzyme (ACE) gene has been associated with a) adverse vascular events contributing to early mortality and b) progressive deterioration of renal function in a variety of chronic glomerular diseases. We investigated the potential role of ACE polymorphisms in patients with systemic lupus erythematosus (SLE). Two hundred and sixteen (216) SLE patients (121 Caucasians; 78 African Americans; and 17 other) and 200 normal controls were studied; 134 patients had evidence of renal disease. ACE genotypes were determined by a polymerase chain reaction based assay. The frequency of genotype DD was increased in African American normal controls compared to Caucasians (55% vs. 37%, p = 0.017) and in African American normal controls vs. African American lupus patients (55% vs. 30%, p = 0.008). Trend analysis of the genotype distribution across the three African American groups (renal, non-renal, controls) revealed a trend of increased frequency of I and decreased frequency of D as likelihood of renal disease increases (p = 0.008). No association between any ACE genotype with parameters of renal disease and/or response to therapy was identified. African American patients with lupus have a lower frequency of DD genotype as compared to African American normal controls. Further studies will be necessary to address whether this is due to decreased survival of these patients, a protective effect of DD genotype from developing the disease or a chance sample effect.