Identification of the gene altered in Berardinelli-Seip congenital lipodystrophy on chromosome 11q13.

Congenital generalized lipodystrophy, or Berardinelli-Seip syndrome (BSCL), is a rare autosomal recessive disease characterized by a near-absence of adipose tissue from birth or early infancy and severe insulin resistance. Other clinical and biological features include acanthosis nigricans, hyperandrogenism, muscular hypertrophy, hepatomegaly, altered glucose tolerance or diabetes mellitus, and hypertriglyceridemia. A locus (BSCL1) has been mapped to 9q34 with evidence of heterogeneity. Here, we report a genome screen of nine BSCL families from two geographical clusters (in Lebanon and Norway). We identified a new disease locus, designated BSCL2, within the 2.5-Mb interval flanked by markers D11S4076 and D11S480 on chromosome 11q13. Analysis of 20 additional families of various ethnic origins led to the identification of 11 families in which the disease cosegregates with the 11q13 locus; the remaining families provide confirmation of linkage to 9q34. Sequence analysis of genes located in the 11q13 interval disclosed mutations in a gene homologous to the murine guanine nucleotide-binding protein (G protein), gamma3-linked gene (Gng3lg) in all BSCL2-linked families. BSCL2 is most highly expressed in brain and testis and encodes a protein (which we have called seipin) of unknown function. Most of the variants are null mutations and probably result in a severe disruption of the protein. These findings are of general importance for understanding the molecular mechanisms underlying regulation of body fat distribution and insulin resistance.

A four-day subrenal capsule assay for testing the effectiveness of anticancer drugs against human tumors.

The subrenal capsule assay may predict to which anticancer drug a given patient's tumor is sensitive and may also be used to screen new anticancer drugs. The present study documents that the use of this model requires a histological assessment of both the exploitability of a subrenal capsule assay and the extent of drug-induced antitumor lesions. Thirty-five tumors from 34 patients with solid tumor were submitted to a subrenal capsule assay in a total of 1130 male B6D2F1 mice. After being biopsied, each tumor was dissected by a pathologist and cut into 50 pieces (1.5 X 1.5 X 1.5 cu mm), and one piece was implanted under the renal capsule of 35 mice; the mean tumor diameter was measured on Day 0. Mice were randomized into groups of 6 to 10 animals each. On Days 1, 2, and 3, mice were treated either with placebo (control group) or with various anticancer agents. On Days 4 or 6, mice were sacrificed, the mean tumor diameter measured, and the tumor-bearing kidney fixed in Bouin's picroformol solution and processed for histological analysis after staining with hematein -eosin. Seven histological parameters were blindly rated in a semiquantitative fashion yielding a compound score ( PAPAN ) which estimated the overall quality of each xenograft between -3 and +11. On Day 4, as opposed to Day 6, mean lymphocytic infiltration was 3-fold lower (p less than 0.01), and the rate of xenografts containing well-preserved cancer cells was 2-fold larger (p less than 0.01) in three different tumor specimens. Twenty-two of 31 (71%) assays were evaluable, as defined by a histological quality control test. In those, drug effects were demonstrable by statistically significant differences among groups in 2 assays (9%) by using the relative variation in tumor size as an index of drug effectiveness and in 12 assays (54%) by PAPAN histological score. This suggests the higher sensitivity of histological scoring over tumor size measurements. Moreover, no correlation between relative variation in tumor size and PAPAN was demonstrable with statistical significance indicating the poor reliability of tumor size measurements as an index of the antitumor effectiveness of cytostatic drugs.

Lamin A/C gene: sex-determined expression of mutations in Dunnigan-type familial partial lipodystrophy and absence of coding mutations in congenital and acquired generalized lipoatrophy.

Missense mutations of the lamin A/C gene, LMNA, have been recently identified in Dunnigan-type familial partial lipodystrophy (FPLD), which belongs to a heterogeneous group of rare disorders affecting adipose tissue distribution and metabolism. In this study, we sequenced the LMNA coding region from patients presenting with FPLD or other forms of lipodystrophy. We identified two heterozygous mutations in exon 8, R482W and R482Q, in FPLD patients (six families and one individual) with various clinical presentations. In addition, we found a novel heterozygous mutation (R584H) in exon 11, encoding specifically the lamin A isoform, in a patient with typical FPLD. Clinical and biochemical investigations in FPLD patients revealed that the expression and the severity of the phenotype were markedly dependent on sex, with female patients being more markedly affected. In subjects with generalized lipoatrophy, either congenital (13 case subjects) or acquired (14 case subjects), or Barraquer-Simon syndrome (2 case subjects), the entire LMNA coding sequence was normal. Although FPLD mutations are predominantly localized in exon 8 of LMNA, the finding of a novel mutation at codon 584, together with the R582H heterozygous substitution recently described, confirms that the C-terminal region specific to the lamin A isoform is a second susceptibility region for mutations in FPLD.

Genetic exclusion of 14 candidate genes in lipoatropic diabetes using linkage analysis in 10 consanguineous families.

Lipoatropic diabetes (LD) is a rare recessive autosomal disorder, mainly characterized by lipoatrophy with alterations in lipid metabolism and extreme insulin resistance. To identify molecular defects responsible for this disease, we tested the implication of 14 candidate genes coding for proteins involved either in insulin action, i.e. insulin receptor, insulin receptor substrate 1, insulin-like growth factor I receptor, diabetes-associated ras-like protein (Rad), and glycogen synthase, or in lipid metabolism, i.e. lipoprotein lipase; apolipoproteins CII, AII, and CIII; hepatic lipase; hormone-sensitive lipase; the beta 3-adrenergic receptor; leptin; and fatty acid-binding protein 2. To this end, haplotype and linkage analyses using genotyping with microsatellites in 10 consanguineous families provided us with powerful genetic tools. Our results show that in most families, lod scores at a null recombination fraction were less than -2. Haplotype analysis also argues against the involvement of these genes in LD. This implies that mutations in these genes are unlikely to make a major genetic contribution to LD.

Cytostatic action of two nitrosoureas derived from cysteamine.

2-Chloroethyl nitrosocarbamoylcystamine or ICIG-1325 (CNCC) is a lipid-soluble isomeric mixture of nitrosoureas. Its dose-effect relationship on L1210 leukaemia is characterized by a large maximally efficient dose-range (MEDR), greater than that of other nitrosoureas. CNCC also demonstrated significant therapeutic activity on intracerebrally (i.c.) transplanted L1210 leukaemia and on six transplanted solid tumours, TM2 mammary carcinoma, M555 ovarian carcinoma, B16 melanoma, glioma 26, 3LL, Lewis lung carcinoma and colon 26 carcinoma. It was inactive on fibrosarcoma ICIG-Ci4. Its antitumour activity spectrum is wider than that of the related compounds 2-[3-(2-chloroethyl) 3-nitrosoureido]D-glucopyranose (CZT), (chloro-2-ethyl)-1(ribofuranosyl-isopropylidene-2'-3' paranitrobenzoate-5')-3 nitrosourea (RFCNU), and (chloro-2-ethyl)-1 (ribopyranosyl triacetate-2'-3'-4')-3 nitrosourea (RPCNU). A study of its metabolic disposition in animals has shown that CNCC undergoes extensive first-pass metabolism leading to the formation of four main plasma metabolites. These metabolites are water-soluble nitrosoureas that arose from the bioreduction of the disulphide bridge followed by the methylation and the oxidation of the thiol groups. Experimental screening was performed with these chemically synthesized metabolites. Both N'-(2-chloroethyl)-N-[2-(methylsulphinyl)ethyl]-N'-nitrosourea (CMSOEN2) and N'-(2-chloroethyl)-N-[2-(methylsulphonyl)ethyl]-N'-nitrosourea (CMSO2EN2) are very active on L1210 leukaemia grafted intraperitoneally (i.p.) and i.c., L40 leukaemia, B16 melanoma, glioma 26 and Lewis lung carcinoma. Their effectiveness is better than that of the parent compound CNCC. In addition,the percentage of mice cured after CMSOEN2 or CMSO2EN2 treatment is increased especially on B16 melanoma and glioma 26. 6 Haematological toxicity of both active metabolites is lower than that of CNCC, particularly on platelets which is the main toxicity location due to nitrosoureas.

Experimental antitumor activity of 5'-nor-anhydrovinblastine navelbine.

We have found that navelbine, a new semi-synthetic Vinca alkaloid analogue, is as active as vincristine on implanted murine tumors. It has a low cross-resistance with vincristine and vindesine.

Antitumor activity of l-OHP in mice.

The isomeric mixtures of platinum complexes of diaminocyclohexane (DACH) had been found active on several murine tumors. A recent separation of the oxalato-platinum complex of trans-l-DACH isomer allowed more precise screening studies and permitted the selection of one compound: l-OHP was submitted to our murine tumor screening system. The drug was given: (a) at doses of 1-12 mg/kg i.p. or i.v. on day 1, 5 and 9 compared to identical doses of cis-dichlorodiamine platinum II (CDDP) in L1210 bearing mice and (b) to AkR leukemia, LGC lymphoma, glioma 26, B16 melanoma, MA 16-C mammary carcinoma and Lewis lung carcinoma bearing mice at 2 dosages: 5 mg/kg (minimal effective dose on L1210), and 8 mg/kg (subtoxic dose in L1210). Acute LD10 and LD50 appeared similar to CDDP and l-OHP. l-OHP administered i.p. was more active on L1210 than CDDP. On L1210 grafted intracerebrally and on LGC lymphoma l-OHP increased significantly the lifespan while CDDP was inactive. On AkR leukemia, both drugs were active but l-OHP was less toxic. Both drugs were inactive on murine solid tumors. No renal toxicity was observed with l-OHP as compared to CDDP.

Experimental protection by a luteinizing hormone-releasing hormone agonist of a bone marrow nitrosourea aplasia. Preliminary observations.

D-Tryptophan-6-luteinizing hormone-releasing hormone (D-Trp6-LH-RH) applied from day -8 to day +15 before and after administration of the nitrosourea analog, N,N'-bis[N-(2-chloroethyl)-N-nitrosocarbamoyl]cysteamine (CNCC), favored bone marrow restoration, as independently evaluated by 3 observers in a double-blind fashion.

Experimental in vivo cross-resistance of vinca alkaloid drugs.

In human therapy, an absence of cross-resistance has been observed between vincristine and vindesine in patients receiving polychemotherapy whilst, in our experimental in vivo studies, such a cross-resistance has been found between Vinca alkaloids. Further studies are required to explain this discrepancy.

Interspersion of cyclophosphamide and BCG in the treatment of L1210 leukemia and Lewis Tumor.

The therapeutic value of interspersing cyclophosphamide (CPM) chemotherapy and BCG immunotherapy was investigated in two tumor models: L1210 leukemia and Lewis solid tumor (LLT). In the case of L1210 leukemia, the antileukemic effect of CPM was enhanced by subsequent BCG administration where a single cycle of combined treatment was applied; treatment by repeated doses of CPM interspersed with BCG was no more effective than CPM chemotherapy alone. In the case of LLT tumor, the effect of one cycle of combined CPM-BCG treatment was not different from CPM administered alone but treatment by repeated doses of CPM interspersed with BCG immunotherapy was less effective than CPM chemotherapy alone. These results indicate that, while the effect of BCG immunotherapy may be favorable or nil when BCG is applied after cell-reducing chemotherapy, it may be nil or unfavorable when applied repeatedly in interspersed chemoimmunotherapy treatments.

Effectiveness of murine leukemia chemotherapy according to the immune state: reconsideration of correlations between chemotherapy, tumour cell killing, and survival time.

Cyclophosphamide (CPM) chemotherapy (134 mg/kg) of L1210 leukemia is less efficient in mice previously immunodepressed by antithymocyte serum (ATS) than in non-ATS pretreated mice. On the other hand, administration of a higher dose of CPM (403 mg/kg), which kills a greater number of leukemic cells but induces an immunodepression, according to the skin graft test, results in a shorter survival time than does the administration of a lower dose of CPM (134 mg/kg), capable of killing fewer leukemic cells but not inducing such an immunodepression. Thus, it appears that: (1) the antileukemic effect of the same dose of a chemotherapeutic drug is less efficient in immunodepressed than in nonimmunodepressed hosts, and (2) calculation of the number of neoplastic cells killed by a given chemotherapy by extrapolation from the survival time may lead to erroneous conclusions.

Ultrastructural study of the cardiotoxicity and light-microscopic findings of the skin after treatment of golden hamsters with seven different anthracyclines.

Golden hamsters were administered seven anthracyclines: adriamycin (ADM), detorubicin (DTR), daunorubicin (DNR), 4'-epi-adriamycin (eADM), rubidazone (RBZ), aclacinomycin (ACM), and N-trifluoroacetyladriamycin-14-valerate (AD-32), three times a week during 4 weeks, at doses equivalent to 3/4 of those which are optimally oncostatic on murine L1210 leukemia. We examined their myocardia by electron microscopy (EM) and their skin by light microscopy (LM), and report here the findings of these two examinations. The mortality was very high for the groups of hamsters treated with ADM, DTR, DRB, eADM, and RBZ (all treated hamsters died before the end of the fourth week) and very low for those treated with ACM and AD-32 (for each drug, only one of the 21 treated animals died after 4 weeks of treatment). After the first week of treatment and chiefly after the second week, all treated hamsters, except those treated with ACM, showed very severe EM alterations of their myocardia. EM detected almost no early myocardial lesions in ACM-treated hamsters but, after 4 weeks of treatment, severe cardiac lesions also appeared which, like those after AD-32, were nonlethal and reversible. LM of the skin detected degenerative lesions with atrophy of all epidermic layers and a loss of the hair (alopecia) in all treated hamsters except those treated with ACM and AD-32; the skin in these two groups preserved its normal histologic structure. These observations agree with phase I-II clinical ACM studies in which the rate of ECG abnormalities was 4.5% and the rate of alopecia 0%, and with an early AD-32 clinical study conducted by Blum [3].

Virostatic in vivo effect of elliptinium on Friend's retrovirus (a model for HIV infection).

As we had discovered the virostatic effects of some analogues of acriflavine, a non-oncostatic intercalating agent, on HIV1 and on its best murine model, Friend's virus [7], we then studied other non- or poorly oncostatic intercalating agents, in particular ellipticins (whose negligible cytostatic effect with methoxy-9-ellipticin we first published. We report in this paper the in vivo virostatic effect of 2-methyl-9-hydroxy-ellipticinium (elliptinium) on Friend's virus.

Can virostatic chemotherapy and/or adoptive allogeneic immunotherapy eradicate in vivo Friend's virus infection?

While none of the three drugs which exert an in vitro anti-HIV effect, neither AZT nor the other two, acriflavine and elliptinium, which we have shown to be more efficient than AZT, is able to eradicate Friend's virus in vivo, the combination of the 3 drugs at much smaller doses than when given alone seems able to eradicate it in almost 30% of the infected animals. This possible eradicating effect of virostatics in combination is compared with the results we had previously obtained with lymphocytes of virus-immunized allogeneic donors.

In vivo eradication of Friend virus as an experimental HIV-model, by combination of zidovudine, acriflavine and an ellipticine analogue. Possible application to the treatment of human pre-AIDS?

Simultaneous administration of zidovudine, acriflavine and celliptium to Friend virus-injected mice eradicates the virus, as evidenced by the impossibility of the treated-mouse serum, when injected to virgin recipients, to induce spleen foci formation. An adapted preliminary protocol given to patients in whose p 24 antigen was present in the blood, lead to a considerable reduction of that marker. The cures lasted 3 weeks, and were repeated after 3-week intervals. Since p 24 antigen returns to pre-treatment levels at the end of the interval, research should concentrate on the maintenance of the effect during the interval.

The in vivo effect of ellipticine analogues on the blood concentration of Friend virus: a murine model for studying anti-HIV drugs.

The viremia of a mouse carrying a murine retrovirus, the Friend virus, was taken as a model for screening drugs which could be active on the human HIV retrovirus. Ellipticine and two of its analogues injected ip at appropriate doses drastically reduced the viremia of the Friend virus-injected mice, as measured by transfer of their serum on day 8 of the treatment to secondary recipients and numeration, in the spleens of the latter, of the foci the remaining virus had induced. Since phase I and II trials have already been published (for leukemia treatment), the efficacy of those drugs on HIV-1 positive AZT-resistant patients can be tested directly.

Comparative microscopic study of cardiotoxicity and skin toxicity of anthracycline analogs.

Golden hamsters were submitted, three times a week during 4 weeks, to i.p. administration of an antracenedione, mitoxantrone (MTX), and 12 different anthracyclines, adriamycin (ADM), detorubicin (DTR), daunorubicin (DNR), 4'-epi-adriamycin (e-ADM), rubidazone (RBZ), aclacinomycin (ACM), N-trifluoroacetyladriamycin-14-valerate (AD-32), tetrahydropyranyl-adriamycin (THP-ADM), N-L-leucyl-daunorubicin (l-DNR), carminomycin (CAM), rubicyclamin (RBC) and N-trifluoroacetyl-adriamycin-14-9-hemiadipate (AD-143), at doses equivalent to 3/4 those which are optimally oncostatic on murine L1210 leukemia. The electron microscopic (EM) study of the myocardium showed that all studied drugs are cardiotoxic, but with different degree of cardiotoxicity. The histopathological study of the skin detect degenerative lesions with different degree of alopecia. According to the degree of their cardiotoxicity, skin toxicity, and general toxicity or mortality, all drugs studied are classified into 3 groups: 1st group, ADM, DNR, 1-DNR and RBZ, causing very severe cardiac alterations and alopecia (grade 2-3), and very high mortality, 2nd group, e-ADM, DTR, CAM RBC and MTX, with less severe cardiac alterations, and alopecia (grade 1-2), and always high mortality, and 3rd group, ACM, THP-ADM, AD-32 and AD-143, causing less severe myocardial alterations (grade 1-2), without alopecia (grade 0), and extremely low mortality and general toxicity.

Effects of B16 melanoma transplantation on the respiration of grouped C57Bl female mice.

Two batches of 110 females C57Bl mice were grafted with B 16 melanoma, the first batch at the age of 67 days, the second at 82 days. The animals were divided into groups of 10, and kept under a LD 12 : 12 regimen. The growth of the tumors was more rapid in the second batch. This was accompanied in this batch, by a decrease in body growth, a diminution of heart rate and an increase in QRSII amplitude. Significant decreases in carbon dioxide emission (VCO2) level, mostly during the dark period of time, when the mice had their greatest activity, and decrease in respiratory photic variations at light transitions were obtained when the tumors were well developed. Harmonic analysis of the continuous recording of carbon dioxide shows slight changes in VCO2 ultradian (40 minutes less than tau less than 12 hours) rhythms. The effects on respiration were confirmed by different survival rates and rectal temperatures between tumor grafted and controls submitted to an acute nitrogen normobaric hypoxia.

Can neo-adjuvant chemotherapy prevent residual tumors?

MA 16/C is a spontaneous mouse mammary adenocarcinoma. It is hormone-dependent and was injected s.c. into C3H/He female mice on day 0. Tumors were excised on day 15. Neo-adjuvant treatments were applied from day 1 to day 21 for hormonotherapy and immunotherapy and on days 1, 5 and 9 for chemotherapy. Adjuvant treatments were applied from day 21 to day 42 for hormonotherapy and immunotherapy, and on days 21, 25 and 29 for chemotherapy. Mixed (neo-adjuvant and adjuvant) treatments combined the two patterns. Chemotherapy consisted of an oxalato-platinum complex of trans-l-dach (l-OHP) at a dose of 5 mg/kg i.p. Hormonotherapy consisted of the LH-RH agonist (D-Trp6) LH-RH, at a dose of 100 micrograms/kg i.p. Zinc gluconate (6mg/kg per os) and bestatin (6mg/kg per os) were administered as immunoregulators. Under present experimental conditions, surgery alone did not increase the life span. Both neo-adjuvant and adjuvant chemotherapy and neo-adjuvant hormonotherapy, however, when added to surgery, increased survival significantly (p less than 0.02-p less than 0.03).